Cyanine
Encyclopedia
Cyanine is a non-systematic name of a synthetic dye family belonging to polymethine
group. Cyanines have many uses as fluorescent dyes, particularly in biomedical imaging. Depending on the structure, they cover the spectrum
from IR
to UV
.
Cyanines were originally used, and still are, to increase the sensitivity range of photographic emulsions, i.e., to increase the range of wavelength
s which will form an image on the film. Cyanines are also used in CD-R
and DVD-R
media. The ones used are mostly green or light blue in color, and are chemically unstable. This makes unstabilized cyanine discs unsuitable for archival CD and DVD use, as they can fade and become unreadable in a few years, however, recent cyanine discs contain stabilizers that slow down the deterioration significantly. These discs are often rated with an archival life of 75 years or more. The other dyes used in CD-Rs are phthalocyanine
and azo
.
Cyanines were first synthesized over a century ago, and there are a large number reported in the literature.
where two nitrogen
s are joined by a polymethine
chain. Both nitrogens are each independently part of a heteroaromatic moiety, such as pyrrole
, imidazole
, thiazole
, pyridine
, quinoline
, indole
, benzothiazole
, etc.
s or protein
molecules. Labeling is done for visualization and quantification purposes. They are used in a wide variety of biological applications including comparative genomic hybridization
and in gene chips, which are used in transcriptomics. They are also used to label protein
s and nucleic acid for various studies including proteomics
and RNA localization.
of the molecule. The Cy3 and Cy5 nomenclature was first proposed by Ernst, et al. in 1989, and is non-standard, since it gives no hint of their chemical structures. In the original paper the number designated the count of the methine
s (as shown), and the side chains were unspecified. Thus various structures are designated Cy3 and Cy5 in the literature.
The R groups
do not have to be identical. In the dyes as used they are short aliphatic chains one or both of which ends in a highly reactive moieties such as N-hydroxysuccinimide
or maleimide
.
The scanners actually use different laser
emission wavelengths (typically 532 nm and 635 nm) and filter wavelengths (550-600 nm and 655-695 nm) to avoid background contamination. They are thus able to easily distinguish between two samples when one sample has been labeled with Cy3 and the other labeled with Cy5. They are also able to quantify the amount of labeling in either sample.
, Dylight
, IRIS Dyes, Seta dyes, SeTau dyes, SRfluor dyes and Square dyes dyes can be used interchangeably with Cy dyes in most biochemical applications.
that microarrays which used Cy5 were susceptible to intermittent data quality decrease caused by environmental ozone. Exposures to ozone levels above 5-10 ppb for 10–30 seconds were reported to decrease the reproducibility of Cy5 microarrays. Much higher levels of ozone (>100 ppb) were required to observe an effect in Cy3.
experiments DNA
or RNA
is labeled with either Cy3 or Cy5 that has been synthesized to carry an N-hydroxysuccinimidyl ester (NHS-ester) reactive group. Since, NHS-esters react readily only with aliphatic amine
groups, which nucleic acids lack, nucleotide
s have to be modified with aminoallyl groups. This is done through incorporating aminoallyl-modified nucleotides during synthesis reactions. A good ratio is a label every 60 bases such that the labels are not too close to each other, thus resulting in quenching
effects.
reactive groups instead. The maleimide functionality allows conjugation of the fluorescent dye to the sulfhydryl group of cysteine
residues. Cysteines can be added and removed from the protein domain of interest via PCR mutagenesis.
Cy5, is sensitive to the electronic environment it resides in. Changes in the conformation of the protein it is attached to will produce an enhancement or quenching of the emission. The rate of this change can be measured to determine enzyme kinetic parameters. The dyes can be used for similar purposes in FRET
experiments.
Cy3 and Cy5 are used in proteomics
experiments so that samples from two sources can be mixed and run together thorough the separation process. This eliminates variations due to differing experimental conditions that are inevitable if the samples were run separately. These variations make it extremely difficult, if not impossible, to use computers to automate the acquisition of the data after the separation is complete. Using these dyes makes the automation trivial.
FOI = C(dye) / C(nucleic acid)
Example: Frequency of Incorporation (FOI) of Cy3 per 1000 bases:
FOI(Cy3) = 58.5 * A550/A260
In multiple biological applications only minimal sample amounts can be afforded for these measurements (e.g. array CGH). Therefore, specialized photometers like the NanoPhotometer™ offer the possibility to determine sample concentrations as well as dye incorporation with submicroliter volumes (starting with 0.3 µl). In addition, due to the reduction of the optical pathlength samples are diluted automatically in comparison to standard cuvette measurements. The respective virtual dilution factors are considered by the software of the instrument. Because the measurements are processed with undiluted samples, the reproducibility of the results is very high. And if desired, samples can be retrieved after the measurement for further processing.
Polymethine
Polymethines are compounds made up from an odd number of methine groups bound together by alternating single and double bonds.Compounds made up from an even number of methine groups are known as polyenes....
group. Cyanines have many uses as fluorescent dyes, particularly in biomedical imaging. Depending on the structure, they cover the spectrum
Spectrum
A spectrum is a condition that is not limited to a specific set of values but can vary infinitely within a continuum. The word saw its first scientific use within the field of optics to describe the rainbow of colors in visible light when separated using a prism; it has since been applied by...
from IR
Infrared
Infrared light is electromagnetic radiation with a wavelength longer than that of visible light, measured from the nominal edge of visible red light at 0.74 micrometres , and extending conventionally to 300 µm...
to UV
Ultraviolet
Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...
.
Cyanines were originally used, and still are, to increase the sensitivity range of photographic emulsions, i.e., to increase the range of wavelength
Wavelength
In physics, the wavelength of a sinusoidal wave is the spatial period of the wave—the distance over which the wave's shape repeats.It is usually determined by considering the distance between consecutive corresponding points of the same phase, such as crests, troughs, or zero crossings, and is a...
s which will form an image on the film. Cyanines are also used in CD-R
CD-R
A CD-R is a variation of the Compact Disc invented by Philips and Sony. CD-R is a Write Once Read Many optical medium, though the whole disk does not have to be entirely written in the same session....
and DVD-R
DVD-R
DVD-R is a DVD recordable format. A DVD-R typically has a storage capacity of 4.71 GB. Pioneer has also developed an 8.5 GB dual layer version, DVD-R DL, which appeared on the market in 2005....
media. The ones used are mostly green or light blue in color, and are chemically unstable. This makes unstabilized cyanine discs unsuitable for archival CD and DVD use, as they can fade and become unreadable in a few years, however, recent cyanine discs contain stabilizers that slow down the deterioration significantly. These discs are often rated with an archival life of 75 years or more. The other dyes used in CD-Rs are phthalocyanine
Phthalocyanine
Phthalocyanine is an intensely blue-green coloured macrocyclic compound that is widely used in dyeing. Phthalocyanines form coordination complexes with most elements of the periodic table...
and azo
Azo compound
Azo compounds are compounds bearing the functional group R-N=N-R', in which R and R' can be either aryl or alkyl. IUPAC defines azo compounds as: "Derivatives of diazene , HN=NH, wherein both hydrogens are substituted by hydrocarbyl groups, e.g. PhN=NPh azobenzene or diphenyldiazene." The more...
.
Cyanines were first synthesized over a century ago, and there are a large number reported in the literature.
Structure
There are three types of cyanines:- Streptocyanines or open chain cyanines:
- R2N+=CH[CH=CH]n-NR2 (I)
- Hemicyanines:
- Aryl=N+=CH[CH=CH]n-NR2 (II)
- Closed chain cyanines:
- Aryl=N+=CH[CH=CH]n-N=Aryl (III)
where two nitrogen
Nitrogen
Nitrogen is a chemical element that has the symbol N, atomic number of 7 and atomic mass 14.00674 u. Elemental nitrogen is a colorless, odorless, tasteless, and mostly inert diatomic gas at standard conditions, constituting 78.08% by volume of Earth's atmosphere...
s are joined by a polymethine
Polymethine
Polymethines are compounds made up from an odd number of methine groups bound together by alternating single and double bonds.Compounds made up from an even number of methine groups are known as polyenes....
chain. Both nitrogens are each independently part of a heteroaromatic moiety, such as pyrrole
Pyrrole
Pyrrole is a heterocyclic aromatic organic compound, a five-membered ring with the formula C4H4NH. It is a colourless volatile liquid that darkens readily upon exposure to air. Substituted derivatives are also called pyrroles, e.g., N-methylpyrrole, C4H4NCH3...
, imidazole
Imidazole
Imidazole is an organic compound with the formula C3H4N2. This aromatic heterocyclic is a diazole and is classified as an alkaloid. Imidazole refers to the parent compound, whereas imidazoles are a class of heterocycles with similar ring structure, but varying substituents...
, thiazole
Thiazole
Thiazole, or 1,3-thiazole, is a heterocyclic compound that contains both sulfur and nitrogen; the term 'thiazole' also refers to a large family of derivatives. Thiazole itself is a pale yellow liquid with a pyridine-like odor and the molecular formula C3H3NS...
, pyridine
Pyridine
Pyridine is a basic heterocyclic organic compound with the chemical formula C5H5N. It is structurally related to benzene, with one C-H group replaced by a nitrogen atom...
, quinoline
Quinoline
Quinoline is a heterocyclic aromatic organic compound. It has the formula C9H7N and is a colourless hygroscopic liquid with a strong odour. Aged samples, if exposed to light, become yellow and later brown...
, indole
Indole
Indole is an aromatic heterocyclic organic compound. It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring. Indole is a popular component of fragrances and the precursor to many pharmaceuticals. Compounds that contain an...
, benzothiazole
Benzothiazole
Benzothiazole is an aromatic heterocyclic compound with the chemical formula . It is colorless, slightly viscous liquid. Although the parent compound, benzothiazole is not widely used, many of its derivatives are found in commercial products or in nature...
, etc.
Cy3 and Cy5
Cy3 and Cy5 are reactive water-soluble fluorescent dyes of the cyanine dye family. Cy3 dyes are red (~550 nm excitation, ~570 nm emission and therefore appear red), while Cy5 is fluorescent in the far red region (~650/670 nm) but absorbs in the orange region (~649 nm). They are usually synthesized with reactive groups on either one or both of the nitrogen side chains so that they can be chemically linked to either nucleic acidNucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
s or protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
molecules. Labeling is done for visualization and quantification purposes. They are used in a wide variety of biological applications including comparative genomic hybridization
Comparative genomic hybridization
Comparative genomic hybridization or Chromosomal Microarray Analysis is a molecular-cytogenetic method for the analysis of copy number changes in the DNA content of a given subject's DNA and often in tumor cells....
and in gene chips, which are used in transcriptomics. They are also used to label protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s and nucleic acid for various studies including proteomics
Proteomics
Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells. The term "proteomics" was first coined in 1997 to make an analogy with...
and RNA localization.
Nomenclature and Structure
Standard chemical names specify exactly the chemical structureChemical structure
A chemical structure includes molecular geometry, electronic structure and crystal structure of molecules. Molecular geometry refers to the spatial arrangement of atoms in a molecule and the chemical bonds that hold the atoms together. Molecular geometry can range from the very simple, such as...
of the molecule. The Cy3 and Cy5 nomenclature was first proposed by Ernst, et al. in 1989, and is non-standard, since it gives no hint of their chemical structures. In the original paper the number designated the count of the methine
Methine
In chemistry, methine is a trivalent functional group CH, derived formally from methane. The methine group consists of a carbon atom bound by two single bonds and one double bond, where one of the single bonds is to a hydrogen...
s (as shown), and the side chains were unspecified. Thus various structures are designated Cy3 and Cy5 in the literature.
The R groups
Side chain
In organic chemistry and biochemistry, a side chain is a chemical group that is attached to a core part of the molecule called "main chain" or backbone. The placeholder R is often used as a generic placeholder for alkyl group side chains in chemical structure diagrams. To indicate other non-carbon...
do not have to be identical. In the dyes as used they are short aliphatic chains one or both of which ends in a highly reactive moieties such as N-hydroxysuccinimide
Succinimide
Succinimide is a cyclic imide with the formula C4H5NO2. It is used in a variety of organic syntheses, as well as in some industrial silver plating processes.-Succinimides:...
or maleimide
Maleimide
Maleimide is the chemical compound with the formula H2C22NH . This unsaturated imide is an important building block in organic synthesis. The name is a contraction of maleic acid and imide, the -CNHC- functional group...
.
Spectral characteristics
| Dye Dye A dye is a colored substance that has an affinity to the substrate to which it is being applied. The dye is generally applied in an aqueous solution, and requires a mordant to improve the fastness of the dye on the fiber.... |
Absorbance Max | Emission Emission Emission may refer to:* Flue gas, also:** Exhaust gas, flue gas occurring as a result of the combustion of a fuel* Emission of air pollutants... Max |
Quantum yield Quantum yield The quantum yield of a radiation-induced process is the number of times that a defined event occurs per photon absorbed by the system. The "event" may represent a chemical reaction, for example the decomposition of a reactant molecule:... in PBS buffer |
Molecular weight (Da) |
|---|---|---|---|---|
| Cy3 | 550 nm | 570 nm | 0.04 | 766 |
| Cy5 | 649 nm | 670 nm | 0.28 | 792 |
The scanners actually use different laser
Laser
A laser is a device that emits light through a process of optical amplification based on the stimulated emission of photons. The term "laser" originated as an acronym for Light Amplification by Stimulated Emission of Radiation...
emission wavelengths (typically 532 nm and 635 nm) and filter wavelengths (550-600 nm and 655-695 nm) to avoid background contamination. They are thus able to easily distinguish between two samples when one sample has been labeled with Cy3 and the other labeled with Cy5. They are also able to quantify the amount of labeling in either sample.
Cy dye alternatives
Alexa Fluor dyes, Atto DyesATTO dyes
ATTO Dyes are a series of fluorescent labels and dyes manufactured by ATTO-TEC GmbH in Siegen, Germany. The dyes have been designed for application in the area of life science, e.g...
, Dylight
DyLight Fluor
The DyLight Fluor family of fluorescent dyes are produced by Dyomics in collaboration with Thermo Fisher Scientific. DyLight dyes are typically used in biotechnology and research applications as biomolecule, cell and tissue labels for fluorescence microscopy, cell biology or molecular...
, IRIS Dyes, Seta dyes, SeTau dyes, SRfluor dyes and Square dyes dyes can be used interchangeably with Cy dyes in most biochemical applications.
Cy5 ozone susceptibility
In 2003, researchers at Inpharmatics and Agilent reported in Analytical ChemistryAnalytical Chemistry (journal)
Analytical Chemistry is a peer-reviewed scientific journal, published since 1929 by the American Chemical Society. It is currently indexed/abstracted in: Chemical Abstracts Service, CABI, EBSCOhost, ProQuest, PubMed, Scopus, and Web of Science...
that microarrays which used Cy5 were susceptible to intermittent data quality decrease caused by environmental ozone. Exposures to ozone levels above 5-10 ppb for 10–30 seconds were reported to decrease the reproducibility of Cy5 microarrays. Much higher levels of ozone (>100 ppb) were required to observe an effect in Cy3.
Nucleic acid labeling
In microarrayMicroarray
A microarray is a multiplex lab-on-a-chip. It is a 2D array on a solid substrate that assays large amounts of biological material using high-throughput screening methods.Types of microarrays include:...
experiments DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
or RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
is labeled with either Cy3 or Cy5 that has been synthesized to carry an N-hydroxysuccinimidyl ester (NHS-ester) reactive group. Since, NHS-esters react readily only with aliphatic amine
Amine
Amines are organic compounds and functional groups that contain a basic nitrogen atom with a lone pair. Amines are derivatives of ammonia, wherein one or more hydrogen atoms have been replaced by a substituent such as an alkyl or aryl group. Important amines include amino acids, biogenic amines,...
groups, which nucleic acids lack, nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
s have to be modified with aminoallyl groups. This is done through incorporating aminoallyl-modified nucleotides during synthesis reactions. A good ratio is a label every 60 bases such that the labels are not too close to each other, thus resulting in quenching
Quenching (fluorescence)
Quenching refers to any process which decreases the fluorescence intensity of a given substance. A variety of processes can result in quenching, such as excited state reactions, energy transfer, complex-formation and collisional quenching. As a consequence, quenching is often heavily dependent on...
effects.
Protein labeling
For protein labeling, Cy3 and Cy5 dyes sometimes bear maleimideMaleimide
Maleimide is the chemical compound with the formula H2C22NH . This unsaturated imide is an important building block in organic synthesis. The name is a contraction of maleic acid and imide, the -CNHC- functional group...
reactive groups instead. The maleimide functionality allows conjugation of the fluorescent dye to the sulfhydryl group of cysteine
Cysteine
Cysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid...
residues. Cysteines can be added and removed from the protein domain of interest via PCR mutagenesis.
Cy5, is sensitive to the electronic environment it resides in. Changes in the conformation of the protein it is attached to will produce an enhancement or quenching of the emission. The rate of this change can be measured to determine enzyme kinetic parameters. The dyes can be used for similar purposes in FRET
Fret
A fret is a raised portion on the neck of a stringed instrument, that extends generally across the full width of the neck. On most modern western instruments, frets are metal strips inserted into the fingerboard...
experiments.
Cy3 and Cy5 are used in proteomics
Proteomics
Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells. The term "proteomics" was first coined in 1997 to make an analogy with...
experiments so that samples from two sources can be mixed and run together thorough the separation process. This eliminates variations due to differing experimental conditions that are inevitable if the samples were run separately. These variations make it extremely difficult, if not impossible, to use computers to automate the acquisition of the data after the separation is complete. Using these dyes makes the automation trivial.
Spectrophotometric quantification of dye incorporation
The amount of each sample as well as the amount of incorporated dyes has to be quantified following nucleic acid or protein labeling and purification of the labeling reaction. To determine the dye incorporation rate, the absorbance reading at the wavelength reported for maximum absorbance of the fluorescence dye is used. The corresponding extinction coefficient of the dye is used in the Lambert-Beer Law to determine the dye concentration (c = A / (e * d)). Comparing these values with the DNA, RNA or protein concentration gives a dye incorporation rate (Frequency of incorporation (FOI)).FOI = C(dye) / C(nucleic acid)
Example: Frequency of Incorporation (FOI) of Cy3 per 1000 bases:
FOI(Cy3) = 58.5 * A550/A260
In multiple biological applications only minimal sample amounts can be afforded for these measurements (e.g. array CGH). Therefore, specialized photometers like the NanoPhotometer™ offer the possibility to determine sample concentrations as well as dye incorporation with submicroliter volumes (starting with 0.3 µl). In addition, due to the reduction of the optical pathlength samples are diluted automatically in comparison to standard cuvette measurements. The respective virtual dilution factors are considered by the software of the instrument. Because the measurements are processed with undiluted samples, the reproducibility of the results is very high. And if desired, samples can be retrieved after the measurement for further processing.
See also
- MerocyanineMerocyanineMerocyanines are a class of fluorescent dyes typified by merocycanine I.These dyes are usually intensely colored and have large extinction coefficients....
- N-HydroxysuccinimideN-HydroxysuccinimideN-Hydroxysuccinimide is a compound with a molecular weight of 115.09 and a melting point of 95 °C.As it is slightly acidic, it is an irritant to skin, eyes and mucous membranes....
- MaleimideMaleimideMaleimide is the chemical compound with the formula H2C22NH . This unsaturated imide is an important building block in organic synthesis. The name is a contraction of maleic acid and imide, the -CNHC- functional group...
- Indocyanine greenIndocyanine greenIndocyanine green is a cyanine dye used in medical diagnostics. It is used for determining cardiac output, hepatic function, and liver blood flow, and for ophthalmic angiography. It has a peak spectral absorption at about 800 nm. These infrared frequencies penetrate retinal layers, allowing ICG...
- J-aggregateJ-aggregateA J-aggregate is a type of dye with an absorption band that shifts to a longer wavelength of increasing sharpness when it aggregates under the influence of a solvent or additive or concentration as a result of supramolecular self-organisation. The dye can be characterized further by a small...