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Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, than the absorbed radiation. A perceptible example of fluorescence occurs when the absorbed radiation is in the ultraviolet region of the electromagnetic spectrum, while the emitted light is in the visible region; this gives the fluorescent substance a distinct color that can only be seen when the substance has been exposed to UV light. Fluorescent materials cease to glow nearly immediately when the radiation source stops, unlike phosphorescent materials, which continue to emit light for some time after.
In chemistry, a reagent or analytical reagent is a substance or compound added to a system to cause a chemical reaction, or test if one occurs. The terms reactant and reagent are often used interchangeably, but reactant specifies a substance consumed in the course of a chemical reaction. Solvents, though involved in the reaction mechanism, are usually not called reactants. Similarly, catalysts are not consumed by the reaction, so they are not reactants. In biochemistry, especially in connection with enzyme-catalyzed reactions, the reactants are commonly called substrates.
In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically. Various labeling techniques such as enzymatic labeling, protein labeling, and genetic labeling are widely utilized. Ethidium bromide, fluorescein and green fluorescent protein are common tags. The most commonly labelled molecules are antibodies, proteins, amino acids and peptides which are then used as specific probes for detection of a particular target.
In organic chemistry, an imine is a functional group or organic compound containing a carbon–nitrogen double bond. The nitrogen atom can be attached to a hydrogen or an organic group (R). The carbon atom has two additional single bonds. Imines are common in synthetic and naturally occurring compounds and they participate in many reactions.
In biochemistry, biotinylation is the process of covalently attaching biotin to a protein, nucleic acid or other molecule. Biotinylation is rapid, specific and is unlikely to disturb the natural function of the molecule due to the small size of biotin. Biotin binds to streptavidin and avidin with an extremely high affinity, fast on-rate, and high specificity, and these interactions are exploited in many areas of biotechnology to isolate biotinylated molecules of interest. Biotin-binding to streptavidin and avidin is resistant to extremes of heat, pH and proteolysis, making capture of biotinylated molecules possible in a wide variety of environments. Also, multiple biotin molecules can be conjugated to a protein of interest, which allows binding of multiple streptavidin, avidin or neutravidin protein molecules and increases the sensitivity of detection of the protein of interest. There is a large number of biotinylation reagents available that exploit the wide range of possible labelling methods. Due to the strong affinity between biotin and streptavidin, the purification of biotinylated proteins has been a widely used approach to identify protein-protein interactions and post-translational events such as ubiquitylation in molecular biology.
In chemistry, quenching refers to any process which decreases the fluorescent intensity of a given substance. A variety of processes can result in quenching, such as excited state reactions, energy transfer, complex-formation and collisions. As a consequence, quenching is often heavily dependent on pressure and temperature. Molecular oxygen, iodine ions and acrylamide are common chemical quenchers. The chloride ion is a well known quencher for quinine fluorescence. Quenching poses a problem for non-instant spectroscopic methods, such as laser-induced fluorescence.
Cyanines, also referred to as tetramethylindo(di)-carbocyanines are a synthetic dye family belonging to the polymethine group. Although the name derives etymologically from terms for shades of blue, the cyanine family covers the electromagnetic spectrum from near IR to UV.
A chiral derivatizing agent (CDA) also known as a chiral resolving reagent, is a chiral auxiliary used to convert a mixture of enantiomers into diastereomers in order to analyze the quantities of each enantiomer present within the mix. Analysis can be conducted by spectroscopy or by chromatography. The use of chiral derivatizing agents has declined with the popularization of chiral HPLC. Besides analysis, chiral derivatization is also used for chiral resolution, the actual physical separation of the enantiomers.
Aminoallyl nucleotide is a nucleotide with a modified base containing an allylamine. They are used in post-labeling of nucleic acids by fluorescence detection in microarray. They are reactive with N-Hydroxysuccinimide ester group which helps attach a fluorescent dye to the primary amino group on the nucleotide. These nucleotides are known as 5-(3-aminoallyl)-nucleotides since the aminoallyl group is usually attached to carbon 5 of the pyrimidine ring of uracil or cytosine. The primary amine group in the aminoallyl moiety is aliphatic and thus more reactive compared to the amine groups that are directly attached to the rings (aromatic) of the bases. Common names of aminoallyl nucleosides are initially abbreviated with aa- or AA- to indicate aminoallyl. The 5-carbon sugar is indicated with or without the lowercase "d" indicating deoxyribose if included or ribose if not. Finally the nitrogenous base and number of phosphates are indicated.
Phthalaldehyde (sometimes also o-phthalaldehyde or ortho-phthalaldehyde, OPA) is the chemical compound with the formula C6H4(CHO)2. It is one of three isomers of benzene dicarbaldehyde, related to phthalic acid. This pale yellow solid is a building block in the synthesis of heterocyclic compounds and a reagent in the analysis of amino acids. OPA dissolves in water solution at pH < 11.5. Its solutions degrade upon UV illumination and exposure to air.
Folin's reagent or sodium 1,2-naphthoquinone-4-sulfonate is a chemical reagent used as a derivatizing agent to measure levels of amines and amino acids. The reagent reacts with them in alkaline solution to produce a fluorescent material that can be easily detected.
Dansyl chloride or 5-(DimethylAmino)Naphthalene-1-SulfonYL chloride is a reagent that reacts with primary amino groups in both aliphatic and aromatic amines to produce stable blue- or blue-green–fluorescent sulfonamide adducts. It can also be made to react with secondary amines. Dansyl chloride is widely used to modify amino acids; specifically, protein sequencing and amino acid analysis. Dansyl chloride may also be denoted DNSC. Likewise, a similar derivative, dansyl amide is known as DNSA.
BODIPY is the technical common name of a chemical compound with formula C
9H
7BN
2F
2, whose molecule consists of a boron difluoride group BF
2 joined to a dipyrromethene group C
9H
7N
2; specifically, the compound 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene in the IUPAC nomenclature. The common name is an abbreviation for "boron-dipyrromethene". It is a red crystalline solid, stable at ambient temperature, soluble in methanol.
Silylation is the introduction of one or more (usually) substituted silyl groups (R3Si) to a molecule. Silylations are core methods for production of organosilicon chemistry. Silanization involves similar methods but usually refers to attachment of silyl groups to solids.
Lanthanide probes are a non-invasive analytical tool commonly used for biological and chemical applications. Lanthanides are metal ions which have their 4f energy level filled and generally refer to elements cerium to lutetium in the periodic table. The fluorescence of lanthanide salts is weak because the energy absorption of the metallic ion is low; hence chelated complexes of lanthanides are most commonly used. The term chelate derives from the Greek word for “claw,” and is applied to name ligands, which attach to a metal ion with two or more donor atoms through dative bonds. The fluorescence is most intense when the metal ion has the oxidation state of 3+. Not all lanthanide metals can be used and the most common are: Sm(III), Eu(III), Tb(III), and Dy(III).
3-(2-furoyl)-quinoline-2-carboxaldehyde (FQ) is a fluorogenic amine labeling dye that is not fluorescent itself, but reacts with primary amines to form fluorescent products. It was first reported in 1990. Cyanide, typically provided via KCN or NaCN salts, is a required co-substrate in the fluorogenic reaction. It has been used for the detection of amines and peptides, largely in CE-SDS, where it is recognized to reach a silver stain-like high sensitivity via laser-induced fluorescence. Once bound to protein the excitation wavelength is 480 nm (blue) and the emission wavelength is ~600 nm (orange).
Colorogenic or colourogenic describes a property of chemical compounds which are initially not colored, but become colored through a chemical reaction, often through an intermolecular covalent reaction that either covalently binds the now colored compound to a target molecule, or through a reaction which leads to a colored non-covalently bound product. Colorogenic reagents are often used for qualitative testing for the presence of chemical functional groups. Colorogenic labeling reagents are sometimes used in analytical chemistry procedures, for example in HPLC or CE to derivative target compounds, however the enhanced sensitivity from the analogous fluorogenic reagents, leads colorogenic reagents to be less frequently used in quantitative applications.
4-Fluoro-7-nitrobenzofurazan (NBD-F) is a fluorogenic, amine labeling dye that is not fluorescent itself, but covalently reacts with secondary or primary amines to form a fluorescently labeled product. It and other fluorogenic benzofurans are used for derivitization in HPLC applications. After the fluorogenic reaction, it can be detected with an excitation wavelength of 470 nm (blue) and an emission wavelength of 530 nm (green), enabling an HPLC limit of detection of 10 fmol.
6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) is a fluorogenic, amine labeling dye that is not fluorescent itself, but covalently reacts with secondary amines to form a fluorescently labeled product. It has a fluorescence excitation wavelength of 250 nm (UV-C), and emission wavelength of 395 nm.
3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) is a fluorogenic amine labeling dye that is not fluorescent itself, but covalently reacts with primary amines to form fluorescent products. It was first reported in 1991. Today, it is largely used in the context of quantifying peptides or proteins. Either cyanide or thiols are required as a co-substrate in the fluorogenic reaction, although thiols also react with & mask the CBQCA aldehyde thereby preventing the fluorogenic reaction against the targeted primary amines. Once bound to protein the excitation wavelength is 465 nm (blue) and the emission wavelength is ~550 nm (green).
References
- ↑ McChesney-Harris, L. L.; Kakodakar, S. V.; Bernstein, S. C.; Stobaugh, J. F. (2012-12-22). "Mechanism Based Design of Amine Fluorogenic Derivatization Reagents: Proof of Concept, Physical–Chemical Characterization and Initial Analytical Derivatization Protocols". Chromatographia. Springer Science and Business Media LLC. 76 (3–4): 117–132. doi:10.1007/s10337-012-2371-9. ISSN 0009-5893. S2CID 95016112.
- ↑ "Fluorogenic". IUPAC GoldBook. IUPAC. 2014. doi: 10.1351/goldbook.FT07379 . Retrieved 10 March 2023.
- ↑ Fanali, Salvatore; Haddad, Paul R.; Poole, Colin; Riekkola, Marja-Liisa (2017). Liquid chromatography. Volume 2, Applications. Amsterdam, Netherlands. ISBN 978-0-12-809344-3. OCLC 992119225.
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