Cyanine is a non-systematic name of a synthetic dye family belonging to
polymethine group. The word cyanin is from the English word “cyan", which
conventionally means a shade of blue-green and is derived from the Greek
“kyanos" which means a somewhat different color: "dark blue".
Cyanines were and are still used in industry, and more recently in
biotechnology. Cyanines have many uses as fluorescent dyes, particularly in
biomedical imaging. Depending on the structure, they cover the spectrum from
IR to UV. 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
Hemicyanines: Aryl=N+=CH[CH=CH]n-NR2
Closed chain cyanines: Aryl=N+=CH[CH=CH]n-N=Aryl
where two nitrogens 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.
Cyanine dyes history, and use in industry
Cyanines were first synthesized over a century ago. They were originally used,
and still are, to increase the sensitivity range of photographic
emulsions, i.e., to increase the range of wavelengths which will form an image
on the film, making the film panchromatic. 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.
Conventional Cyanines dyes used in biotechnology
Cyanines dyes are usually synthesized from 2, 3, 5 or 7 methine structures
with reactive groups on either one or both of the nitrogen side chains so that
they can be chemically linked to either nucleic acids or protein molecules.
Labeling is done for visualization and quantification purposes. Biological
applications include comparative genomic hybridization and gene chips, which are
used in transcriptomics, and various studies in proteomics such as RNA
localization, molecular interaction studies by fluorescence energy transfer
and fluorescent immunoassays. Cyanines dyes are available with
different modifications such as methyl, ethyl or butyl substituant, carboxyl,
acetylmethoxy, and sulfo groups. Ex: Excitation wavelength in nanometers
Em: Emission wavelength in nanometers MW: Molecular weight
QY: Quantum yield Main Cy dyes and their uses
Cyanines(Cy) replace advantageously conventional dyes such as
Fluorescein(FITC) and rhodamines, yielding brighter and more stable
fluorescence. Cy 3 and Cy5 are the most popular, used
typically combined for 2 colors detection.
Cy3 fluoresces greenish yellow, while Cy5 is fluorescent in the red region.
Cy3 can be detected by various fluorometers, imagers, and microscopes
with standard filters for Tetramethylrhodamine. Due to inherently
high extinction coefficient, this dye is also easily detected by naked eye on
gels, and in solution. Cy5 become very popular to replace far red fluorescent
dyes, because of its high extinction coefficient and of its fluorophore
emission maximum in red region, where many CCD detectors have maximum
sensitivity, and biological objects have low background.
The scanners actually use different laser emission wavelengths and filter
wavelengths to avoid background contamination. They are thus able to
easily distinguish colors from Cy3 and from Cy5, and also able to quantify the
amount of Cy3 and Cy5 labeling in one sample.
Other cyanine dyes are useful: Cy3.5 can replace SulfoRhodamine 101.
Cy5.5 is a near-infrared fluorescence-emitting dye.
Cy7 is a near-IR fluor that is invisible to the naked eye. It is used in in vivo
imaging applications, as well as the Cy7.5 dye.
Sulfo–Cyanine dyes bear classically one or two Sulfo groups, rendering the Cy
dye water-soluble, but tri- and quadri-sulfonated forms are available
for even higher hydrosolubility. PEGylation is another modification that
confers hydrophilicity, not only to the dye but also to the labeled conjugate.
= Nomenclature and structure = Standard chemical names specify exactly
the chemical structure 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 methines, 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 moiety
such as N-hydroxysuccinimide or maleimide.
= Cy dye alternatives = Many analogs of standard Cy 2 / 3 / 3.5
/ 5 / 5.5 / 7 / 7.5 dyes were developed, using diverse modification: Alexa Fluor
dyes, Dylight, FluoProbes dyes, Sulfo Cy dyes, Seta dyes, IRIS dyes from Cyanine
Technologies and others can be used interchangeably with Cy dyes in most
biochemical applications, with claimed improvements in solubility,
fluorescence, or photostability. = Cy5 ozone susceptibility =
In 2003, researchers at Inpharmatics and Agilent reported in Analytical Chemistry
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 were required to observe an effect in
Cy3. There are devices that claim to remove ambient ozone levels but they
have not been 3rd party tested. = Applications =
Cyanine dyes are used to label proteins, antibodies, peptides, nucleic acid
probes, and any kind of other biomolecules to be used in a variety of
fluorescence detection techniques: Flow cytometry, Microscopy, Microplate
assays, Microarrays, ... . Nucleic acid labeling
In microarray experiments DNA or RNA is labeled with either Cy3 or Cy5 that has
been synthesized to carry an N-hydroxysuccinimidyl ester reactive
group. Since NHS-esters react readily only with aliphatic amine groups, which
nucleic acids lack, nucleotides 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, which would result in quenching effects.
Protein labeling For protein labeling, Cy3 and Cy5 dyes
sometimes bear typically a succinimidyl group to react with amines, or a
maleimide group to react with a sulfhydryl group of cysteine residues.
Cy5 is sensitive to the electronic environment it resides in. Changes in
the conformation of the protein it is attached to will produce either
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
through 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.
See also Merocyanine
N-Hydroxysuccinimide Maleimide
Indocyanine green J-aggregate
Anthocyanin Phycocyanin
References External links