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Fluorescence is one of two kinds of photoluminescence, the emission of light by a substance that has absorbed light or other electromagnetic radiation. When exposed to ultraviolet radiation, many substances will glow (fluoresce) with colored visible light. The color of the light emitted depends on the chemical composition of the substance. Fluorescent materials generally cease to glow nearly immediately when the radiation source stops. This distinguishes them from the other type of light emission, phosphorescence. Phosphorescent materials continue to emit light for some time after the radiation stops. This difference in timing is a result of quantum spin effects.
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 particle physics, the quantum yield of a radiation-induced process is the number of times a specific event occurs per photon absorbed by the system.
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.
In analytical chemistry, a chiral derivatizing agent (CDA), also known as a chiral resolving reagent, is a derivatization reagent that is a chiral auxiliary used to convert a mixture of enantiomers into diastereomers in order to analyze the quantities of each enantiomer present and determine the optical purity of a sample. Analysis can be conducted by spectroscopy or by chromatography. Some analytical techniques such as HPLC and NMR, in their most commons forms, cannot distinguish enantiomers within a sample, but can distinguish diastereomers. Therefore, converting a mixture of enantiomers to a corresponding mixture of diastereomers can allow analysis. 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.
In chemistry, the term substrate is highly context-dependent. Broadly speaking, it can refer either to a chemical species being observed in a chemical reaction, or to a surface on which other chemical reactions or microscopy are performed.
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.
The term bioorthogonal chemistry refers to any chemical reaction that can occur inside of living systems without interfering with native biochemical processes. The term was coined by Carolyn R. Bertozzi in 2003. Since its introduction, the concept of the bioorthogonal reaction has enabled the study of biomolecules such as glycans, proteins, and lipids in real time in living systems without cellular toxicity. A number of chemical ligation strategies have been developed that fulfill the requirements of bioorthogonality, including the 1,3-dipolar cycloaddition between azides and cyclooctynes, between nitrones and cyclooctynes, oxime/hydrazone formation from aldehydes and ketones, the tetrazine ligation, the isocyanide-based click reaction, and most recently, the quadricyclane ligation.
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.
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. 76 (3–4). Springer Science and Business Media LLC: 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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