Fluorescent tag

{{Short description|Biochemical identification method}} thumb|''S. cerevisiae'' septins revealed with fluorescent microscopy utilizing fluorescent labeling

In molecular biology and biotechnology, a '''fluorescent tag''', also known as a '''fluorescent dye''', '''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.<ref name="Sahoo"/> 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.<ref>{{cite web|url=http://pharmaxchange.info/press/2011/01/fluorescent-labeling-of-biomolecules-with-organic-probes/|title=Fluorescent labeling of biomolecules with organic probes - Presentations - PharmaXChange.info|date=29 January 2011}}</ref>

==History== thumb|120px|Stokes George G thumb|120px|Osamu Shimomura-press conference Dec 06th, 2008-1 The development of methods to detect and identify biomolecules has been motivated by the ability to improve the study of molecular structure and interactions. Before the advent of fluorescent labeling, radioisotopes were used to detect and identify molecular compounds. Since then, safer methods have been developed that involve the use of fluorescent dyes or fluorescent proteins as tags or probes as a means to label and identify biomolecules.<ref name="Gwynne and Page">{{cite web|last=Gwynne and Page|first=Peter and Guy|title=Laboratory Technology Trends: Fluorescence + Labeling|url=http://www.sciencemag.org/site/products/labtech.xhtml|publisher=Science|access-date=10 March 2013}}</ref> Although fluorescent tagging in this regard has only been recently utilized, the discovery of fluorescence has been around for a much longer time.

Sir George Stokes developed the Stokes Law of Fluorescence in 1852 which states that the wavelength of fluorescence emission is greater than that of the exciting radiation. Richard Meyer then termed fluorophore in 1897 to describe a chemical group associated with fluorescence. Since then, Fluorescein was created as a fluorescent dye by Adolph von Baeyer in 1871 and the method of staining was developed and utilized with the development of fluorescence microscopy in 1911.<ref name="pmid19233914">{{cite journal | vauthors = Kricka LJ, Fortina P | title = Analytical ancestry: "firsts" in fluorescent labeling of nucleosides, nucleotides, and nucleic acids | journal = Clinical Chemistry | volume = 55 | issue = 4 | pages = 670–83 | date = April 2009 | pmid = 19233914 | doi = 10.1373/clinchem.2008.116152 | doi-access = free }}</ref>

Ethidium bromide and variants were developed in the 1950s,<ref name="pmid19233914"/> and in 1994, fluorescent proteins or FPs were introduced.<ref name="urlChemical Tags for Labeling Proteins Inside Living Cells">{{cite journal | vauthors = Jing C, Cornish VW | title = Chemical tags for labeling proteins inside living cells | journal = Accounts of Chemical Research | volume = 44 | issue = 9 | pages = 784–92 | date = September 2011 | pmid = 21879706 | pmc = 3232020 | doi = 10.1021/ar200099f }}</ref> Green fluorescent protein or GFP was discovered by Osamu Shimomura in the 1960s and was developed as a tracer molecule by Douglas Prasher in 1987.<ref name="urlGreen Fluorescent Protein - GFP History - Osamu Shimomura">{{cite web |url=http://www.conncoll.edu/ccacad/zimmer/GFP-ww/shimomura.html |title=Green Fluorescent Protein - GFP History - Osamu Shimomura }}</ref> FPs led to a breakthrough of live cell imaging with the ability to selectively tag genetic protein regions and observe protein functions and mechanisms.<ref name="urlChemical Tags for Labeling Proteins Inside Living Cells"/> For this breakthrough, Shimomura was awarded the Nobel Prize in 2008.<ref name=NobelPrize>{{cite web|last=Shimomura|first=Osamu|title=The Nobel Prize in Chemistry|url=https://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/press.html|access-date=5 April 2013}}</ref>

New methods for tracking biomolecules have been developed including the use of colorimetric biosensors, photochromic compounds, biomaterials, and electrochemical sensors. Fluorescent labeling is also a common method in which applications have expanded to enzymatic labeling, chemical labeling, protein labeling, and genetic labeling.<ref name=Sahoo>{{cite journal|last=Sahoo|first=Harekrushna|title=Fluorescent labeling techniques in biomolecules: a flashback|journal=RSC Advances|date=1 January 2012|volume=2|issue=18|pages=7017–7029|doi=10.1039/C2RA20389H|bibcode=2012RSCAd...2.7017S}}</ref>

450px|thumbnail|Types of biosensors

==Methods for tracking biomolecules== There are currently several labeling methods for tracking biomolecules. Some of the methods include the following.

===Isotope markers=== Common species that isotope markers are used for include proteins. In this case, amino acids with stable isotopes of either carbon, nitrogen, or hydrogen are incorporated into polypeptide sequences.<ref name="Mass Tagging Chen">{{cite journal | vauthors = Chen X, Smith LM, Bradbury EM | title = Site-specific mass tagging with stable isotopes in proteins for accurate and efficient protein identification | journal = Analytical Chemistry | volume = 72 | issue = 6 | pages = 1134–43 | date = March 2000 | pmid = 10740850 | doi = 10.1021/ac9911600 }}</ref> These polypeptides are then put through mass spectrometry. Because of the exact defined change that these isotopes incur on the peptides, it is possible to tell through the spectrometry graph which peptides contained the isotopes. By doing so, one can extract the protein of interest from several others in a group. Isotopic compounds play an important role as photochromes, described below.

===Colorimetric biosensors=== Biosensors are attached to a substance of interest. Normally, this substance would not be able to absorb light, but with the attached biosensor, light can be absorbed and emitted on a spectrophotometer.<ref name=colorassay>{{cite web|title=Colorimetric Assays|url=http://www.ruf.rice.edu/~bioslabs/methods/protein/protcurve.html|access-date=3 April 2013}}</ref> Additionally, biosensors that are fluorescent can be viewed with the naked eye. Some fluorescent biosensors also have the ability to change color in changing environments (ex: from blue to red). A researcher would be able to inspect and get data about the surrounding environment based on what color he or she could see visibly from the biosensor-molecule hybrid species.<ref name="Biosensor Vesicles Revital">{{cite journal|last=Halevy, Revital|author2=Sofiya Kolusheval |author3=Robert E.W. Hancock |author4=Raz Jelinek |title=Colorimetric Biosensor Vesicles for Biotechnological Applications|journal=Materials Research Society Symposium Proceedings.| volume = 724. Biological and Biomimetic Materials - Properties to Function|year=2002|url=http://apps.dtic.mil/dtic/tr/fulltext/u2/p014413.pdf|archive-url=https://web.archive.org/web/20131014173930/http://www.dtic.mil/dtic/tr/fulltext/u2/p014413.pdf|url-status=live|archive-date=October 14, 2013|access-date=4 April 2013}}</ref>

Colorimetric assays are normally used to determine how much concentration of one species there is relative to another.<ref name=colorassay />

===Photochromic compounds=== Photochromic compounds have the ability to switch between a range or variety of colors. Their ability to display different colors lies in how they absorb light. Different isomeric manifestations of the molecule absorbs different wavelengths of light, so that each isomeric species can display a different color based on its absorption. These include photoswitchable compounds, which are proteins that can switch from a non-fluorescent state to that of a fluorescent one given a certain environment.<ref name="Lummer Photoswitchable">{{cite journal | vauthors = Lummer M, Humpert F, Wiedenlübbert M, Sauer M, Schüttpelz M, Staiger D | title = A new set of reversibly photoswitchable fluorescent proteins for use in transgenic plants | journal = Molecular Plant | volume = 6 | issue = 5 | pages = 1518–30 | date = September 2013 | pmid = 23434876 | doi = 10.1093/mp/sst040 | doi-access = free }}</ref>

The most common organic molecule to be used as a photochrome is diarylethene.<ref name="Perrier photochrome">{{cite journal | vauthors = Perrier A, Maurel F, Jacquemin D | title = Single molecule multiphotochromism with diarylethenes | journal = Accounts of Chemical Research | volume = 45 | issue = 8 | pages = 1173–82 | date = August 2012 | pmid = 22668009 | doi = 10.1021/ar200214k }}</ref> Other examples of photoswitchable proteins include PADRON-C, rs-FastLIME-s and bs-DRONPA-s, which can be used in plant and mammalian cells alike to watch cells move into different environments.<ref name="Lummer Photoswitchable" />

===Biomaterials=== Fluorescent biomaterials are a possible way of using external factors to observe a pathway more visibly. The method involves fluorescently labeling peptide molecules that would alter an organism's natural pathway. When this peptide is inserted into the organism's cell, it can induce a different reaction. This method can be used, for example to treat a patient and then visibly see the treatment's outcome.<ref name="Zhang Biomaterials">{{cite journal | vauthors = Zhang Y, Yang J | title = Design Strategies for Fluorescent Biodegradable Polymeric Biomaterials | journal = Journal of Materials Chemistry B | volume = 1 | issue = 2 | pages = 132–148 | date = January 2013 | pmid = 23710326 | pmc = 3660738 | doi = 10.1039/C2TB00071G }}</ref>

===Electrochemical sensors=== Electrochemical sensors can be used for label-free sensing of biomolecules. They detect changes and measure current between a probed metal electrode and an electrolyte containing the target analyte. A known potential to the electrode is then applied from a feedback current and the resulting current can be measured. For example, one technique using electrochemical sensing includes slowly raising the voltage causing chemical species at the electrode to be oxidized or reduced. Cell current vs voltage is plotted which can ultimately identify the quantity of chemical species consumed or produced at the electrode.<ref name="urlbioee.ee.columbia.edu">{{cite web |url=http://bioee.ee.columbia.edu/downloads/2007/7.pdf |title=bioee.ee.columbia.edu |archive-url=https://web.archive.org/web/20121220222500/http://www.bioee.ee.columbia.edu/downloads/2007/7.pdf |archive-date=2012-12-20 |url-status=dead }}</ref> Fluorescent tags can be used in conjunction with electrochemical sensors for ease of detection in a biological system.

===Fluorescent labels=== thumb|200px|''Aequorea victoria'' thumb|200px|GFP structure

Of the various methods of labeling biomolecules, fluorescent labels are advantageous in that they are highly sensitive even at low concentration and non-destructive to the target molecule folding and function.<ref name="Sahoo"/>

Green fluorescent protein is a naturally occurring fluorescent protein from the jellyfish ''Aequorea victoria'' that is widely used to tag proteins of interest. GFP emits a photon in the green region of the light spectrum when excited by the absorption of light. The chromophore consists of an oxidized tripeptide -Ser^65-Tyr^66-Gly^67 located within a β barrel. GFP catalyzes the oxidation and only requires molecular oxygen. GFP has been modified by changing the wavelength of light absorbed to include other colors of fluorescence. YFP or yellow fluorescent protein, BFP or blue fluorescent protein, and CFP or cyan fluorescent protein are examples of GFP variants. These variants are produced by the genetic engineering of the GFP gene.<ref name="isbn0-7167-7108-X">{{cite book |author1=Cox, Michael |author2=Nelson, David R. |author3=Lehninger, Albert L |title=Lehninger principles of biochemistry |publisher=W.H. Freeman |location=San Francisco |year=2008 |isbn=978-0-7167-7108-1 |url-access=registration |url=https://archive.org/details/lehningerprincip00lehn_1 }}</ref>

Synthetic fluorescent probes can also be used as fluorescent labels. Advantages of these labels include a smaller size with more variety in color. They can be used to tag proteins of interest more selectively by various methods including chemical recognition-based labeling, such as utilizing metal-chelating peptide tags, and biological recognition-based labeling utilizing enzymatic reactions.<ref name="pmid23318293">{{cite journal | vauthors = Jung D, Min K, Jung J, Jang W, Kwon Y | title = Chemical biology-based approaches on fluorescent labeling of proteins in live cells | journal = Molecular BioSystems | volume = 9 | issue = 5 | pages = 862–72 | date = May 2013 | pmid = 23318293 | doi = 10.1039/c2mb25422k }}</ref> However, despite their wide array of excitation and emission wavelengths as well as better stability, synthetic probes tend to be toxic to the cell and so are not generally used in cell imaging studies.<ref name="Sahoo"/>

Fluorescent labels can be hybridized to mRNA to help visualize interaction and activity, such as mRNA localization. An antisense strand labeled with the fluorescent probe is attached to a single mRNA strand, and can then be viewed during cell development to see the movement of mRNA within the cell.<ref name="urlMaking the message clear: visualizing mRNA localization">{{cite journal | vauthors = Weil TT, Parton RM, Davis I | title = Making the message clear: visualizing mRNA localization | journal = Trends in Cell Biology | volume = 20 | issue = 7 | pages = 380–90 | date = July 2010 | pmid = 20444605 | pmc = 2902723 | doi = 10.1016/j.tcb.2010.03.006 }}</ref>

=== Fluorogenic labels === A fluorogen is a ligand (fluorogenic ligand) which is not itself fluorescent, but when it is bound by a specific protein or RNA structure becomes fluorescent.<ref>{{cite journal | vauthors = Szent-Gyorgyi C, Schmidt BF, Schmidt BA, Creeger Y, Fisher GW, Zakel KL, Adler S, Fitzpatrick JA, Woolford CA, Yan Q, Vasilev KV, Berget PB, Bruchez MP, Jarvik JW, Waggoner A | title = Fluorogen-activating single-chain antibodies for imaging cell surface proteins | journal = Nature Biotechnology | volume = 26 | issue = 2 | pages = 235–40 | date = February 2008 | pmid = 18157118 | doi = 10.1038/nbt1368 | s2cid = 21815631 | type = Abstract | quote = We report here the development of protein reporters that generate fluorescence from otherwise dark molecules (fluorogens). }}</ref>

For instance, FAST is a variant of photoactive yellow protein which was engineered to bind chemical mimics of the GFP tripeptide chromophore.<ref>{{cite journal | vauthors = Plamont MA, Billon-Denis E, Maurin S, Gauron C, Pimenta FM, Specht CG, Shi J, Quérard J, Pan B, Rossignol J, Moncoq K, Morellet N, Volovitch M, Lescop E, Chen Y, Triller A, Vriz S, Le Saux T, Jullien L, Gautier A | title = Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 113 | issue = 3 | pages = 497–502 | date = January 2016 | pmid = 26711992 | pmc = 4725535 | doi = 10.1073/pnas.1513094113 | bibcode = 2016PNAS..113..497P | doi-access = free }}</ref> Likewise, the spinach aptamer is an engineered RNA sequence which can bind GFP chromophore chemical mimics, thereby conferring conditional and reversible fluorescence on RNA molecules containing the sequence.<ref name="pmid21798953">{{cite journal | vauthors = Paige JS, Wu KY, Jaffrey SR | title = RNA mimics of green fluorescent protein | journal = Science | volume = 333 | issue = 6042 | pages = 642–6 | date = July 2011 | pmid = 21798953 | pmc = 3314379 | doi = 10.1126/science.1207339 | bibcode = 2011Sci...333..642P }}</ref>

Fluorescent labeling is known for its non-destructive nature and high sensitivity. This has made it one of the most widely used methods for labeling and tracking biomolecules.<ref name="Sahoo"/> Several techniques of fluorescent labeling can be utilized depending on the nature of the target.

===Enzymatic labeling=== In enzymatic labeling, a DNA construct is first formed, using a gene and the DNA of a fluorescent protein.<ref name=Biotechniques>{{cite journal | vauthors = Richter A, Schwager C, Hentze S, Ansorge W, Hentze MW, Muckenthaler M | title = Comparison of fluorescent tag DNA labeling methods used for expression analysis by DNA microarrays | journal = BioTechniques | volume = 33 | issue = 3 | pages = 620–8, 630 | date = September 2002 | pmid = 12238772 | doi = 10.2144/02333rr05 | url = http://www.biotechniques.com/multimedia/archive/00011/02333rr05_11834a.pdf | doi-access = free }}</ref> After transcription, a hybrid RNA + fluorescent is formed. The object of interest is attached to an enzyme that can recognize this hybrid DNA. Usually fluorescein is used as the fluorophore.

===Chemical labeling=== Chemical labeling or the use of chemical tags utilizes the interaction between a small molecule and a specific genetic amino acid sequence.<ref name="pmid21567974">{{cite journal | vauthors = Wombacher R, Cornish VW | title = Chemical tags: applications in live cell fluorescence imaging | journal = Journal of Biophotonics | volume = 4 | issue = 6 | pages = 391–402 | date = June 2011 | pmid = 21567974 | doi = 10.1002/jbio.201100018 | doi-access = free }}</ref> Chemical labeling is sometimes used as an alternative for GFP. Synthetic proteins that function as fluorescent probes are smaller than GFP's, and therefore can function as probes in a wider variety of situations. Moreover, they offer a wider range of colors and photochemical properties.<ref name="chemical labelling Jung">{{cite journal | vauthors = Jung D, Min K, Jung J, Jang W, Kwon Y | title = Chemical biology-based approaches on fluorescent labeling of proteins in live cells | journal = Molecular BioSystems | volume = 9 | issue = 5 | pages = 862–72 | date = May 2013 | pmid = 23318293 | doi = 10.1039/C2MB25422K }}</ref> With recent advancements in chemical labeling, Chemical tags are preferred over fluorescent proteins due to the architectural and size limitations of the fluorescent protein's characteristic β-barrel. Alterations of fluorescent proteins would lead to loss of fluorescent properties.<ref name="pmid21567974"/>

===Protein labeling=== Protein labeling use a short tag to minimize disruption of protein folding and function. Transition metals are used to link specific residues in the tags to site-specific targets such as the N-termini, C-termini, or internal sites within the protein. Examples of tags used for protein labeling include biarsenical tags, Histidine tags, and FLAG tags.<ref name="Sahoo"/>

===Genetic labeling=== Fluorescence in situ hybridization (FISH), is an example of a genetic labeling technique that utilizes probes that are specific for chromosomal sites along the length of a chromosome, also known as chromosome painting. Multiple fluorescent dyes that each have a distinct excitation and emission wavelength are bound to a probe which is then hybridized to chromosomes. A fluorescence microscope can detect the dyes present and send it to a computer that can reveal the karyotype of a cell. This technique allows abnormalities such as deletions and duplications to be revealed.<ref name="isbn1-4292-3413-X">{{cite book |author1=Matthew P Scott |author2=Lodish, Harvey F. |author3=Arnold Berk |author4=Kaiser, Chris |author5=Monty Krieger |author6=Anthony Bretscher |author7=Hidde Ploegh |author8=Angelika Amon |title=Molecular Cell Biology |publisher=W. H. Freeman |location=San Francisco |year=2012 |isbn=978-1-4292-3413-9 }}</ref>

===Analytical chemistry=== {{expert|Chemistry|date=March 2026}} [[File:Rhodamine 123.svg|thumbnail|Rhodamine, a fluorescent molecule often used in small molecule sensors]] '''Small-molecule sensors''' is jargon for chemicals that detect certain '''metal ions''' in solution.<ref name="Zn Sensors (Lippard)1">{{cite journal|last1=Tomat|first1=Elisa|last2=Lippard|first2=Stephen J|title=Imaging mobile zinc in biology|journal=Current Opinion in Chemical Biology|date=April 2010|volume=14|issue=2|pages=225–230|doi=10.1016/j.cbpa.2009.12.010|pmid=20097117|pmc=2847655}}</ref> Although many types exist, most small molecule sensors comprise a subunit that selectively binds to a metal that in turn induces a change in a fluorescent subunit. This change can be observed in the small molecule sensor's spectrum, which can be monitored using a detection system such as a microscope or a photodiode.<ref name="New fluorescent chemosensors for metal ions in solution">{{cite journal|last1=Formica|first1=Mauro|last2=Fusi|first2=Vieri|last3=Giorgi|first3=Luca|last4=Micheloni|first4=Mauro|title=New fluorescent chemosensors for metal ions in solution|journal=Coordination Chemistry Reviews|date=January 2012|volume=256|issue=1–2|pages=170–192|doi=10.1016/j.ccr.2011.09.010}}</ref> Different probes exist for a variety of applications, each with different dissociation constants with respect to a particular metal, different fluorescent properties, and sensitivities. They probe biological processes by monitoring metal ions at low concentrations in biological systems. More traditional bio-sensing are less effective or not suitable.<ref name="Synthetic fluorescent sensors for studying the cell biology of metals">{{cite journal|last1=Domaille|first1=Dylan W|last2=Que|first2=Emily L|last3=Chang|first3=Christopher J|title=Synthetic fluorescent sensors for studying the cell biology of metals|journal=Nature Chemical Biology|date=March 2008|volume=4|issue=3|pages=168–175|doi=10.1038/nchembio.69|pmid=18277978}}</ref> Most detection mechanisms involved in small molecule sensors involve fluorescence.<ref name="New fluorescent chemosensors for metal ions in solution" /><ref name="Fluorescent Sensors for Measuring Metal Ions in Living Systems" />

====Mechanisms of detection==== 400px|thumbnail|Cartoon depicting a shift in spectrum of a small molecule sensor upon the binding of a metal

*Paramagnetic Fluorescence Quenching, the allowance of new electronic states upon binding a paramagnetic metal atom<ref name="New fluorescent chemosensors for metal ions in solution" /> *Photoinduced Electron Transfer (PET), the blocking of a lower energy state due to the binding of a metal atom.<ref name="New fluorescent chemosensors for metal ions in solution" /> *Photoinduced Charge Transfer (PCT), the modulation of energy levels in a complex by charge transfer within a conjugated pi system.<ref name="New fluorescent chemosensors for metal ions in solution" /> *[http://chemwiki.ucdavis.edu/Theoretical_Chemistry/Fundamentals/Fluorescence_Resonance_Energy_Transfer Fluorescence Resonance Energy Transfer (FRET)],<ref name="New fluorescent chemosensors for metal ions in solution" /> the transfer of an exciton from a donor to an acceptor, modulating the emission spectrum.<ref name="New fluorescent chemosensors for metal ions in solution" /><ref name="Chemwiki Forster">{{cite web|title=Fluorescence Resonance Energy Transfer|url=http://chemwiki.ucdavis.edu/Theoretical_Chemistry/Fundamentals/Fluorescence_Resonance_Energy_Transfer |website=UC Davis Chemwiki|date=2 October 2013 |publisher=UC Davis|access-date=12 March 2015}}</ref> *Excimer/Exciplex formation, the formation of a state that is a hybrid of the ground and excited states. This has novel fluorescent properties.<ref name="New fluorescent chemosensors for metal ions in solution" /> *Chemodosimeters, complexes that undergo irreversible reactions with other species upon binding a metal to form new compounds with novel fluorescent spectra.<ref name="New fluorescent chemosensors for metal ions in solution" />

Fluorophores are essential to some measurement of the metal binding event, and indirectly, metal concentration. There are many types, all with different properties that make them advantageous for different applications. Some work as small metal sensors completely on their own while others must be complexed with a subunit that can chelate or bind a metal ion. Rhodamine for example undergoes a conformation change upon the binding of a metal ion. In so doing it switches between a colorless, non-fluorescent spirocyclic form to a fluorescent, pink open cyclic form.<ref name="New fluorescent chemosensors for metal ions in solution" /><ref name="Aminoxy-linked rhodamine hydroxamate as fluorescent chemosensor for Fe3+ in aqueous media">{{cite journal |last1=Moon |first1=Kyung-Soo |last2=Yang |first2=Young-Keun |last3=Ji |first3=Seunghee |last4=Tae |first4=Jinsung |title=Aminoxy-linked rhodamine hydroxamate as fluorescent chemosensor for Fe3+ in aqueous media |journal=Tetrahedron Letters |date=June 2010 |volume=51 |issue=25 |pages=3290–3293 |doi=10.1016/j.tetlet.2010.04.068}}</ref> Quinoline based sensors have been developed that form luminescent complexes with Cd(II) and fluorescent ones with Zn(II). It is hypothesized to function by changing its lowest luminescent state from n–{{pi}}* to {{pi}}–{{pi}}* when coordinating to a metal.<ref name="New fluorescent chemosensors for metal ions in solution" /><ref name="The synthesis of azacrown ethers with quinoline-based sidearms as potential zinc(II) fluorophores">{{cite journal |last1=Xue |first1=Guoping |last2=Bradshaw |first2=Jerald S |last3=Dalley |first3=N.Kent |last4=Savage |first4=Paul B |last5=Izatt |first5=Reed M |last6=Prodi |first6=Luca |last7=Montalti |first7=Marco |last8=Zaccheroni |first8=Nelsi |title=The synthesis of azacrown ethers with quinoline-based sidearms as potential zinc(II) fluorophores |journal=Tetrahedron |date=June 2002 |volume=58 |issue=24 |pages=4809–4815 |doi=10.1016/S0040-4020(02)00451-9}}</ref><ref name="Time-Dependent DFT Study of Emission Mechanism of 8-Hydroxyquinoline Derivatives as Fluorescent Chemosensors for Metal Ions">{{cite journal |last1=Miyamoto |first1=Ryo |last2=Kawakami |first2=Jun |last3=Takahashi |first3=Shuko |last4=Ito |first4=Shunji |last5=Nagaki |first5=Masahiko |last6=Kitahra |first6=Haruo |title=Time-Dependent DFT Study of Emission Mechanism of 8-Hydroxyquinoline Derivatives as Fluorescent Chemosensors for Metal Ions |journal=Journal of Computer Chemistry, Japan |date=2006 |volume=5 |issue=1 |pages=19–22 |doi=10.2477/jccj.5.19|doi-access=free }}</ref> When the Dansyl group DNS binds to a metal, it loses a sulfonamide hydrogen, causing fluorescence quenching via a PET or reverse PET mechanism in which an electron is transferred either to or from the metal that is bound.<ref name="An Anthracene-Based Fluorescent Sensor for Transition Metal Ions">{{cite journal |last1=Fabbrizzi |first1=Luigi |last2=Licchelli |first2=Maurizio |last3=Pallavicini |first3=Piersandro |last4=Perotti |first4=Angelo |last5=Sacchi |first5=Donatella |title=An Anthracene-Based Fluorescent Sensor for Transition Metal Ions |journal=Angewandte Chemie International Edition in English |date=17 October 1994 |volume=33 |issue=19 |pages=1975–1977 |doi=10.1002/anie.199419751}}</ref>

Small molecule sensors for zinc have been reported.<ref name="Fluorescent Sensors for Measuring Metal Ions in Living Systems">{{cite journal|last1=Carter|first1=Kyle P.|last2=Young|first2=Alexandra M.|last3=Palmer|first3=Amy E.|title=Fluorescent Sensors for Measuring Metal Ions in Living Systems|journal=Chemical Reviews|date=23 April 2014|volume=114|issue=8|pages=4564–4601|doi=10.1021/cr400546e|pmid=24588137|pmc=4096685}}</ref> One example is "ZX1", a compound comprising a dipicolylamine (DPA) Zinc binding subunit that has greater affinity for Zinc than other species found in solution such as Ca and Mg.<ref name="Vesicular Zinc (McNamara)">{{cite journal|last1=Pan|first1=Enhui|last2=Zhang|first2=Xiao-an|last3=Huang|first3=Zhen|last4=Krezel|first4=Artur|last5=Zhao|first5=Min|last6=Tinberg|first6=Christine E.|last7=Lippard|first7=Stephen J.|last8=McNamara|first8=James O.|title=Vesicular Zinc Promotes Presynaptic and Inhibits Postsynaptic Long-Term Potentiation of Mossy Fiber-CA3 Synapse|journal=Neuron|date=September 2011|volume=71|issue=6|pages=1116–1126|doi=10.1016/j.neuron.2011.07.019|pmid=21943607|pmc=3184234}}</ref> <ref name=" Two-Photon Fluorescent Chemosensors Based on the GFP-Chromophore for the Detection of Zn2+ in Biological Samples – From Design to Application ">{{cite journal|last1=Csomos|first1=Attila|last2=Kovacs|first2=Ervin|last3=Madarasz|first3=Miklos|last4=Fedor|first4=Flora|last5=Fulop|first5=Anna|last6=Katona|first6=Gergely|last7=Balazs J.|first7=Rozsa|last8=Mucsi|first8=Zoltan|title=Two-Photon Fluorescent Chemosensors Based on the GFP-Chromophore for the Detection of Zn2+ in Biological Samples – From Design to Application|journal=Sensors and Accuators B|date=1 January 2024|volume=398|issue=1|article-number=134753|doi=10.1016/j.snb.2023.134753 |doi-access=free}}{{Creative Commons text attribution notice|cc=by3|from this source=yes}}</ref> GFZnP OMe is an alternate, GFP-based fluorescent Zn2+ sensor is published for two-photon microscopy and related biological and microscop application. It composed of an 8-methoxyquinoline scaffold. It has excellent photophysical characteristics including a 37-fold fluorescence enhancement with l(ex) = 440 nm and l(em) = 505 nm. The two-photon cross-section is as high as 73 GM at 880 nm.<ref name=" A GFP inspired 8-methoxyquinoline-derived fluorescent molecular sensor for the detection of Zn2+ by two-photon microscopy ">{{cite journal|last1=Csomos|first1=Attila|last2=Madarasz|first2=Miklos|last3=Turczel|first3=Gábor|last4=Levente|first4=cseri|last5=Bodor|first5=Andrea|last6=Matuscsak|first6=Anett|last7=Katona|first7=Gergely|last8=Kovacs|first8=Ervin|last9=Rozsa|first9=Balazs J.|last10=Mucsi|first10=Zoltan|title= A GFP inspired 8-methoxyquinoline-derived fluorescent molecular sensor for the detection of Zn2+ by two-photon microscopy|journal= Chemistry: A European Journal|date=3 June 2024|volume=30|issue=31|article-number=e202400009|doi=10.1002/chem.202400009|pmid=38446718 |bibcode=2024ChEuJ..30E0009C |doi-access=free}}</ref> GFZnP BIPY features a 2,2'-bipyridine chelator moiety. It was effective at physiologically relevant pH-range and excellent photophysical characteristics are reported, including a 53-fold fluorescence enhancement with excitation and emission maxima at 422 nm and 492 nm, respectively. High two-photon cross-section of 3.0 GM at 840 nm as well as excellent metal ion selectivity are reported. In vitro experiments on HEK 293 cell culture were carried out using two-photon microscopy demonstrating the applicability.<ref name=" A molecular hybrid of the GFP chromophore and 2,2′-bipyridine: an accessible sensor for Zn2+ detection with fluorescence microscopy ">{{cite journal |last1=Csomos |first1=Attila |last2=Madarász |first2=Miklós |last3=Turczel |first3=Gábor |last4=Cseri |first4=Levente |last5=Katona |first5=Gergely |last6=Rózsa |first6=Balázs |last7=Kovács |first7=Ervin |last8=Mucsi |first8=Zoltán |display-authors=5 |title=A Molecular Hybrid of the GFP Chromophore and 2,2′-Bipyridine: An Accessible Sensor for Zn2+ Detection with Fluorescence Microscopy |journal=International Journal of Molecular Sciences |date=January 2024 |volume=25 |issue=6 |page=3504 |doi=10.3390/ijms25063504 |doi-access=free |pmid=38542479 |pmc=10971390 |language=en |issn=1422-0067}}{{Creative Commons text attribution notice|cc=by4|from this source=yes}}</ref>

For copper, the CTAP-1 sensor shows a response in the UV region when Cu(I) binds to an azatetrathiacrown motif that in turn excites a pyrazoline-based dye that is attached.<ref name="Synthetic fluorescent sensors for studying the cell biology of metals" /><ref name="Fluorescent Sensors for Measuring Metal Ions in Living Systems" /> In Coppersensor-1 (CS1), a thioether-rich motif binds to Cu(I) causing the excitation of a boron-dipyrromethene (BODIPY) dye in the visible region.<ref name="Synthetic fluorescent sensors for studying the cell biology of metals" /><ref name="Fluorescent Sensors for Measuring Metal Ions in Living Systems" />

Iron sensors include Pryrene-TEMPO, in which the binding of iron to TEMPO quenches the fluorescence of pyrene when no Fe(II) is bound. Upon binding however, TEMPO is reduced and pyrene regains fluorescence. This probe is limited in that an analogous response can be generated by unwanted free radicals, and that it can only by used in acidic solution.<ref name="Fluorescent Sensors for Measuring Metal Ions in Living Systems" /><ref name="High selective determination iron(II) by its enhancement effect on the fluorescence of pyrene-tetramethylpiperidinyl (TEMPO) as a spin fluorescence probe">{{cite journal|last1=Chen|first1=Jin-Long|last2=Zhuo|first2=Shu-Juan|last3=Wu|first3=Yu-Qing|last4=Fang|first4=Fang|last5=Li|first5=Ling|last6=Zhu|first6=Chang-Qing|title=High selective determination iron(II) by its enhancement effect on the fluorescence of pyrene-tetramethylpiperidinyl (TEMPO) as a spin fluorescence probe|journal=Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy|date=February 2006|volume=63|issue=2|pages=438–443|doi=10.1016/j.saa.2005.04.057|pmid=15996513|bibcode=2006AcSpA..63..438C}}</ref> The DansSQ Fe(II)-binding system consists of a Dansyl group bound to styrylquinoline and operates by the disruption of intra-molecular charge transfer. It is limited in that it is only soluble in acetonitrile in 10% H<sub>2</sub>O.<ref name="Fluorescent Sensors for Measuring Metal Ions in Living Systems" />

Cobalt sensors have been made that capitalize on the breaking of C-O bonds by Co(II) in a fluorescent probe known as Cobalt Probe 1 (CP1).<ref name="Co Fluorescent Probe (Chang)">{{cite journal|last1=Au-Yeung|first1=Ho Yu|last2=New|first2=Elizabeth J.|last3=Chang|first3=Christopher J.|title=A selective reaction-based fluorescent probe for detecting cobalt in living cells|journal=Chemical Communications|date=2012|volume=48|issue=43|pages=5268–70|doi=10.1039/c2cc31681a|pmid=22531796}}</ref>

thumbnail|300px|A Sodium-Potassium pump that causes changing concentrations of metal ions in a biological system. Potential applications can be envisioned for detecing mercury in fish.<ref name="Screening Mercury Levels in Fish with a Selective Fluorescent Chemosensor">{{cite journal|last1=Yoon|first1=Sungho|last2=Albers|first2=Aaron E.|last3=Wong|first3=Audrey P.|last4=Chang|first4=Christopher J.|title=Screening Mercury Levels in Fish with a Selective Fluorescent Chemosensor|journal=Journal of the American Chemical Society|date=November 2005|volume=127|issue=46|pages=16030–16031|doi=10.1021/ja0557987|pmid=16287282|bibcode=2005JAChS.12716030Y }}</ref> Some mercury sensors (MS) are complexes of fluorescein and napthofluorescein. The MS1 probe increases its emission upon binding of Hg(II), while maintaining great affinity for mercury over other heavy metal ions.<ref name="Synthetic fluorescent sensors for studying the cell biology of metals" /> The S3 sensor is based on a BODIPY complex which undergoes a significant increase in fluorescence upon the binding of Hg(II).<ref name="Synthetic fluorescent sensors for studying the cell biology of metals" /><ref name="A Highly Selective and Sensitive Fluorescent Chemosensor for Hg in Neutral Buffer Aqueous Solution">{{cite journal|last1=Guo|first1=Xiangfeng|last2=Qian|first2=Xuhong|last3=Jia|first3=Lihua|title=A Highly Selective and Sensitive Fluorescent Chemosensor for Hg in Neutral Buffer Aqueous Solution|journal=Journal of the American Chemical Society|date=March 2004|volume=126|issue=8|pages=2272–2273|doi=10.1021/ja037604y|pmid=14982408|bibcode=2004JAChS.126.2272G }}</ref> MF1 uses a soft thioether chelator for Hg(II) bound to a fluorescein-like xanthenone reporter. It has good contrast upon binding mercury and good selectivity. MF1 is sensitive enough that it has been proposed to be used to test fish for toxic levels of mercury.<ref name="Synthetic fluorescent sensors for studying the cell biology of metals" /><ref name="Screening Mercury Levels in Fish with a Selective Fluorescent Chemosensor" />

==Cell imaging== Chemical tags have been tailored for imaging technologies more so than fluorescent proteins because chemical tags can localize photosensitizers closer to the target proteins.<ref>{{cite book |last1=Ettinger |first1=A |title=Quantitative Imaging in Cell Biology |chapter=Fluorescence live cell imaging |series=Methods in Cell Biology |date=2014 |volume=123|pages=77–94 |doi=10.1016/B978-0-12-420138-5.00005-7 |pmid=24974023 |pmc=4198327 |isbn=9780124201385 }}</ref> Proteins can then be labeled and detected with imaging such as super-resolution microscopy, Ca<sup>2+</sup>-imaging, pH sensing, hydrogen peroxide detection, chromophore assisted light inactivation, and multi-photon light microscopy. In vivo imaging studies in live animals have been performed for the first time with the use of a monomeric protein derived from the bacterial haloalkane dehalogenase known as the Halo-tag.<ref name="pmid21567974"/><ref name="pmid23115610">{{cite journal | vauthors = N Peterson S, Kwon K | title = The HaloTag: Improving Soluble Expression and Applications in Protein Functional Analysis | journal = Current Chemical Genomics | volume = 6 | issue = 1 | pages = 8–17 | year = 2012 | pmid = 23115610 | pmc = 3480702 | doi = 10.2174/1875397301206010008 }}</ref> The Halo-tag covalently links to its ligand and allows for better expression of soluble proteins.<ref name="pmid23115610"/>

==Advantages== Although fluorescent dyes may not have the same sensitivity as radioactive probes, they are able to show real-time activity of molecules in action.<ref name=FlourescentAdvantages>{{cite journal | vauthors = Proudnikov D, Mirzabekov A | title = Chemical methods of DNA and RNA fluorescent labeling | journal = Nucleic Acids Research | volume = 24 | issue = 22 | pages = 4535–42 | date = November 1996 | pmid = 8948646 | pmc = 146275 | doi = 10.1093/nar/24.22.4535 }}</ref> Moreover, radiation and appropriate handling is no longer a concern.

With the development of fluorescent tagging, fluorescence microscopy has allowed the visualization of specific proteins in both fixed and live cell images. Localization of specific proteins has led to important concepts in cellular biology such as the functions of distinct groups of proteins in cellular membranes and organelles. In live cell imaging, fluorescent tags enable movements of proteins and their interactions to be monitored.<ref name="isbn1-4292-3413-X"/>

Latest advances in methods involving fluorescent tags have led to the visualization of mRNA and its localization within various organisms. Live cell imaging of RNA can be achieved by introducing synthesized RNA that is chemically coupled with a fluorescent tag into living cells by microinjection. This technique was used to show how the ''oskar'' mRNA in the ''Drosophila'' embryo localizes to the posterior region of the oocyte.<ref name="urlMaking the message clear: visualizing mRNA localization"/>

==Further reading== *<ref>{{cite journal |last1=Han |first1=Junyan |last2=Burgess |first2=Kevin |title=Fluorescent Indicators for Intracellular pH |journal=Chemical Reviews |date=2010 |volume=110 |issue=5 |pages=2709–2728 |doi=10.1021/cr900249z |pmid=19831417 }}</ref> *<ref>{{cite journal |last1=Martínez-Máñez |first1=Ramón |last2=Sancenón |first2=Félix |title=Fluorogenic and Chromogenic Chemosensors and Reagents for Anions |journal=Chemical Reviews |date=2003 |volume=103 |issue=11 |pages=4419–4476 |doi=10.1021/cr010421e |pmid=14611267 }}</ref> <ref>{{cite journal |last1=Kim |first1=Ha Na |last2=Ren |first2=Wen Xiu |last3=Kim |first3=Jong Seung |last4=Yoon |first4=Juyoung |title=Fluorescent and colorimetric sensors for detection of lead, cadmium, and mercury ions |journal=Chem. Soc. Rev. |date=2012 |volume=41 |issue=8 |pages=3210–3244 |doi=10.1039/c1cs15245a |pmid=22184584 }}</ref>

==Notes== {{Reflist|33em}}

== External links == {{Library resources box |onlinebooks=no |by=no |lcheading=Fluorescence spectroscopy |label=Fluorescence spectroscopy}} {{Authority control}}

{{DEFAULTSORT:Fluorescent Tag}} Category:Molecular biology Category:Fluorescence techniques