Fluorescent Dye

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Fluorescent dyes are defined as compounds which both absorb and emit
strongly in the visible region, and which owe their potential for application
to their intense fluorescence properties.

From: Handbook of Textile and Industrial Dyeing, 2011

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Fluorescent dyes
R.M. Christie, in Handbook of Textile and Industrial Dyeing, 2011

17.4.1 Fluorescent dyes for textiles

Fluorescent dyes are not considered by the Colour Index as a separate,
identifiable dye application class. Instead, the commercially important
textile fluorescent dyes, which are relatively few in number, are distributed
among the traditional application classes. Thus the methods used to dye
the fibres, yarns or fabrics are characteristic of the particular class, details
of which the reader will find in the relevant chapters of this textbook. The
earliest commercial exploitation of fluorescent dyes was the use of
rhodamines and related products to dye silk with unusually brilliant
colours. Only a very limited number of fluorescent dyes reported as
suitable for application to natural protein and cellulosic fibres are
described in the Colour Index. C.I. Acid Yellow 7 (7a) and C.I. Direct Yellow 96
(15) are well-established dyes. However, they are not extensively used on
textiles, one reason being inferior light fastness. They are more suited to
applications such as hydrogeological tracing. C.I. Acid Yellow 250 (structure
not disclosed) is reported as useful for dyeing the felt for yellow tennis
balls. There is one reactive dye of the vinylsulphone type (C.I. Reactive
Yellow 78, 7c) reported in the Colour Index, although described as having no
current commercial supplier. Interestingly, a fluorescent dye, Remazol
Luminous Yellow FL, was introduced by DyStar in 2002. It was claimed as
the first fluorescent reactive dye for cellulosic fibres suitable for high-
visibility workwear and sportswear applications. It is reported as being

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compatible in application with other Remazol reactive dyes, and especially
useful for dyeing fluorescent yellow shades on blends of cellulosics with
polyester and polyamides (Michel, 2002).

Fluorescent dyes are of much greater importance for application to

synthetic fibres such as polyester, polyamides and polyacrylonitrile,
sometimes also in conjunction with elastane fibres. The most important
textile applications are on polyester and thus the most important
fluorescent textile dyes are from the disperse dye class. Fluorescent
disperse dyes are also used, but to a lesser extent, on polyamides and
cellulose acetates. Most of the commercial dyes are yellow, providing the
very familiar greenish-yellow fluorescent colours very commonly
encountered on garments worn for safety purposes. There are also a
number of oranges and reds. The important fluorescent coumarin dyes
include C.I. Disperse Yellows 82 (2a), 184 (2d), 186 (3) and 232 (2d), and C.I.
Disperse Reds 277 (5a) and 374 (5b). An interesting publication analyses
the market for fluorescent dyes in Poland and assesses the suitability of
the commercially available yellow dyes to meet the requirements of the
European standard (EN 471:1997) for high-visibility ‘warning’ clothing
(Szuster et al., 2004). The paper also describes an analytical investigation
which suggests that C.I. Disperse Yellow 184:1 is chemically identical to C.I.
Disperse Yellow 232 (2d). This is a particularly important commercial dye,
providing brilliant greenish-yellow shades on polyester with good fastness
to light, sublimation and washing (Ayyanger et al., 1990). Apparently, dye 3,
usually referred to as C.I. Disperse Yellow 186, also has a duplicate
designation as C.I. Disperse Yellow 202. The range of fluorescent dyes
disclosed in the Colour Index also includes the aminonaphthalimides, C.I.
Disperse Yellows 11 (6a) and 199 (8), and Orange 32 (9), the
benzothioxanthone, C.I. Disperse Reds 303 (mixture of 12 and 13) and the
heterocyclic C.I. Disperse Yellow 139 (17). There are a number of water-
soluble fluorescent cationic dyes of commercial significance for application
to acrylic fibres, including the coumarin C.I. Basic Yellow 40 (6a) and the
methine dyes C.I. Basic Red 13 (16a) and C.I. Basic Violet 7 (16b).
Fluorescent disperse dyes on polyester and cationic dyes on acrylic fibres
show technical performance characteristics typical of the dye application
class. The commercial fluorescent dyes recommended for textiles are often
criticised for light fastness properties which are unable to meet the
standards required by more demanding applications. However, this feature
can be improved by judicious use of light stabilising additives such as UV
absorbers.

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Synthetic Biology, Part A

Gürol Süel, in Methods in Enzymology, 2011

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1.2 Fluorescent dyes
Fluorescent dyes, also known as reactive dyes or fluorophores, have been
used by biologists for decades. Fluorescent dyes offer higher photostability
and brightness compared to fluorescent proteins and do not require a
maturation time. However, fluorescent dyes are usually targeted to
proteins of interest by antibody conjugates or peptide tags. This requires
fixation of cells, which renders measurement of genetic circuit dynamics
impossible. Several fluorescent dyes can be used in living cells, but in many
cases their applicability is still limited. The remainder of this chapter will
focus on the use of fluorescent proteins as the reporter of choice, but
many discussion points also apply to the use of fluorescent dyes.

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Gel Electrophoresis
A. Drabik, ... J. Silberring, in Proteomic Profiling and Analytical Chemistry
(Second Edition), 2016

7.2.8 Fluorescent Dyes Used in Difference Gel Electrophoresis

Fluorescent dyes used for protein separation on the same gel have to meet
several conditions:

•
They must possess the same MW and charge to assure that the same
proteins stained with different dyes will be found at the same position on
the gel.

•
They must replace the charge characteristic for the amino acid residue to
which they are bound.

•
They must possess a different range of absorption and emission, which
makes possible the observation of different proteins labeled with different
fluorescent dyes.

There are three cyanine dyes used in DIGE (Table 7.2.2): Cy2, Cy3 and Cy5.
They possess a broad dynamic range (more than 3.6 orders of magnitude)
and are characterized by linearity and sensitivity (for minimal labeling: Cy2:
0.075 ng; Cy3: 0.025 ng; Cy5: 0.025 ng; and for saturation labeling: below
15 pg). Proteins labeled with Cy dyes may be analyzed and identified by
mass spectrometry.

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Six-membered Rings with One Heteroatom and Fused
Carbocyclic Derivatives
Graham R. Geen, ... Antonio K. Vong, in Comprehensive Heterocyclic Chemistry
II, 1996

5.09.7.1 Fluorescent Dyes and Brighteners

Fluorescent dyes have been defined as materials that both absorb and emit
strongly in the visible region of the spectrum, thereby distinguishing them
from the fluorescent brighteners which emit visible light but absorb in the
ultraviolet. Reviews are available specific to the former 〈93MI 509-08〉
and latter 〈84DP109, 87MI 509-03〉 categories.

Coumarins are one of the most commercially significant groups of

fluorescent dyes and brighteners, most of which are 3,7-disubstituted. The
dyes are characterized by the presence of electron-releasing substituents
at the 7-position, and electron-withdrawing (often heterocyclic)
substituents at the 3-position. Examples include the red dyes (108;
R = alkyl, X = NH, O or S), and the greenish-yellow (109), all used for dyeing
polyester.

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Coumarin fluorescent whiteners counteract the short wavelength visible

light absorption of fibres, paper or plastics by having fluorescence maxima
at 430–440 nm. The hydroxycoumarins formerly used have now been
replaced by compounds having nitrogen-containing substituents at both
the 3- and 7-positions. These are frequently heterocyclic in nature (e.g.
110). Both nonionic coumarins for whitening polyester and water-soluble
cationic coumarins for whitening polyacrylonitrile are available.

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The usefulness of coumarins as fluorescent

whiteners is limited by their susceptibility to
photo-dimerization at the 3,4-position. All four

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products corresponding to the syn- and anti-isomers of the head-to-head
(111) and head-to-tail (112) dimers are normally found, and all are
nonfluorescent (Equation (1)). The major photochemical reactions of
coumarin dyes have been reviewed 〈92UK1243〉.

(1)

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Peptide Fragmentation/Deletion Side Reactions

Yi Yang, in Side Reactions in Peptide Synthesis, 2016

1.7 Acidolysis of peptides with N-terminal FITC modification

Fluorescent dyes are nowadays routinely utilized as labeling compounds
for biomacromolecules. They have been intensively utilized in domains
such as fluorescence microscope, flow cytometry, immunofluorescence
techniques, and so forth. FITC is one of the most frequently employed
fluorescent dyes that could function selectively with amino21 and/or
sulfhydryl22 functional groups in peptides or proteins, visualizing by this
means the affected peptides/proteins under fluorescence. Modifications of
target peptides by FITC could be realized in the process of SPPS23,24
following selective liberation of the amino groups on Lys or Orn side
chains,25 or alternatively, on peptide backbone N α groups.

Side reaction resembling Edman degradation might take place during FITC-
mediated modification on peptide N α functional group. 26 Edman
degradation, as an intentional method for peptide sequencing, is achieved
by the function of the N α group from the target peptide/protein with
phenylisothiocynate, and the subsequent acidolysis of the generated
phenylthiocarbamoyl derivative into a degraded peptide with a deletion
sequence and a split phenylthiohydantoin compound.27 Peptide modified
at its N α functional group by FITC could undergo an equivalent process
upon TFA treatment as well. The thiocarbamoyl derivative 23 derived from
the reaction between the target peptide and FITC is firstly transformed into
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a 5-member ring intermediate 24 under acidic condition, that is
subsequently split from the parental peptide in the form of fluorescein
thiazolinone 25, and finally rearranged to a stable fluorescein
thiohydantoin compound 26. The mechanism of this process is illustrated
in Fig. 1.11. It could be inferred from the proposed mechanism that
formation of the 5-member ring intermediate 24 is the key step of the
whole process. The spatial proximity between the nitrogen atom from FITC
moiety and the amide carbon of the first amino acid on peptide N-terminus
plays a crucially important role in dictating the propensity of the subjected
peptide to undergo the concerned acidolysis side reaction. Compound 23
will be highly susceptible to the transformation into the corresponding 5-
member ring intermediate 24, provided that no spacer is incorporated
between FITC moiety and the N-terminus of the referred peptide chain.
The subsequent fragmentation process will be thus facilitated.

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Figure 1.11. Mechanism of acidolysis of peptide with N-terminal FITC
modification.

According to the result obtained from a systematic study,26 the

aforementioned degradation process does not take place during FITC-
mediated peptide N-terminal modification, but at the step of peptide-FITC
adduct treatment by TFA. Identical with Edman degradation this side
reaction is catalyzed by acid. Normally a spacer like ɛ-Ahx is squeezed
between FITC and the N-terminus of the target peptide in an effort to
circumvent the occurrence of the undesired acidolysis side reaction by
rationally increasing the distance between the nucleophilic nitrogen on
FITC and the potentially labile amide bond on the peptide backbone,
disfavoring by this means the formation of the stable 5-member ring
intermediate, and thus suppressing the acidolytic side reaction reminiscent
of Edman degradation.

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Oxygen Sensing
Reinhard Dirmeier, ... Robert O. Poyton, in Methods in Enzymology, 2004

Use of Fluorescent Dyes

The fluorescent dyes fluorescein, rhodamine, and their derivatives are
frequently used to measure levels of oxidative stress in mammalian cells15
and yeast.16–19 Although these fluorescent dyes may be useful for
measuring overall levels of oxidative stress, they must be used with care
and the proper controls because they are known to react with and become
oxidized by a variety of compounds besides the reactive oxygen species
(superoxide and hydrogen peroxide) they are assumed to measure. These
other compounds include reduced hemoproteins,20 peroxynitrite, 21
horseradish peroxidase,22,23 Fe2+ , Fe2+ -H2O,22 and tyrosine. 24 In addition,
oxidation of these dyes is sensitive to the levels of the antioxidants
catalase,15,22 superoxide dismutase,22 and glutathione,23 and fluorescein
oxidation increases with the pH of the growth medium.23

We have used the fluorescent dye carboxy-2′,7′-dichlorofluorescein

diacetate (carboxy-H 2DCFDA) to measure steady-state ROS levels in both
respiratory-proficient and respiratory-deficient yeast strains, in yeast cells
cultured both aerobically and anaerobically, and in yeast cells shifted from
aerobic to anaerobic conditions.9 Once carboxy-H2DCFDA enters the cell,
the acetate group becomes hydrolyzed by intracellular esterases. The dye
can then become oxidized to the fluorescent product, carboxy-
dichlorofluorescein, which, when excited by light of 500 nm, can be
detected at a wavelength of 525 nm with a spectrofluorometer, fluorescent
microscope, or flow cytometer. Carboxy-DCF has two negative charges at a
physiological pH and thus is thought to be retained within the cell longer
than the uncarboxylated form of the dye. However, carboxy-H2DCFDA is
subject to the same difficulties as described earlier and its oxidation may
be limited by the activity of intracellular esterases.24 We have found that
carboxy-H2DCFDA leaks out of the cell, like its uncarboxylated form
(H 2DCFDA), making the interpretation of long-term, time-dependent
experiments difficult.9,23,25 Consequently, we believe that this dye is useful
for assessing ROS levels in cells grown under steady-state conditions but
not during shift experiments between different oxygen concentrations.

To use carboxy-H2DCFDA for estimating differences in ROS levels between

strains grown in different steady-state conditions or in comparing ROS
levels in mutant and wild-type cells, the dye is dissolved in dimethyl
sulfoxide and added to the culture at a final concentration of 10 μM at least
1 h before measurements are taken. Fluorescence is measured in three
100-μl aliquots, which are diluted 10-fold in 50 mM NaPO4, pH 7.0, and are
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sonicated briefly to disperse the cells. The fluorescence (wavelength, 515–
545 nm) of 5000 cells from each aliquot is then measured using a BD
PharMingen fluorescent-activated cell sorter equipped with a 15-mW argon
laser.

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Single Molecule Tools: Fluorescence Based Approaches,

Part A
Jaime J. Benítez, ... Peng Chen, in Methods in Enzymology, 2010

3.2.3 FRET differentiation of acceptor-blinked/bleached states

from the dissociated state of protein interactions
Organic fluorescent dyes show blinking behavior, that is, the fluorescence
intensity sometimes switches off temporarily. Although fluorescence
blinking can be suppressed significantly by using an oxygen scavenging
system and triplet quenchers (e.g., Trolox; Rasnik et al., 2006), occasional
blinking of the FRET acceptor is problematic, as it would result in an
apparently low FRET efficiency (EFRET = IA/(IA + ID), where IA and ID are the
acceptor and donor fluorescence intensities), which could be mistaken as
that of the dissociated state of protein–protein interactions. Fortunately,
using nanovesicle trapping and Cy3–Cy5 as the FRET pair, the Cy5-blinked
state has clearly lower EFRET than that of the dissociated state from control
experiments (Benitez et al., 2008, 2009).

As far as the apparent EFRET is concerned, the acceptor-blinked state is

effectively the same as that in the absence of the acceptor and that of the
acceptor photobleached state. Therefore, the apparent EFRET from
nanovesicles that merely contain a donor molecule serves as a control for
signal from the acceptor blinked state (Fig. 4.3A). The determined apparent
EFRET with one Cy3 only is 0.04 ± 0.05, which is the same as Cy5-
blinked/bleached state of a Cy3–Cy5 pair (Fig. 4.3C).

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Figure 4.3. smFRET
control experiments
for acceptor
blinked/bleached
states and the
dissociated state. (A)
Two-color
fluorescence intensity
trajectories of a
nanovesicle containing
a single Cy3 molecule
using 532-nm laser
excitation. The Cy3
molecule
photobleaches at the
~ 62th second. (B)
Two-color
fluorescence intensity
trajectories of a
nanovesicle containing
a single Cy3 and a single Cy5. The 532-nm laser is on throughout; the 637-
nm laser was turned on at the ~ 75th second. The Cy3 photobleaches at
the ~ 25th second; the Cy5 molecule photobleaches at ~ 125th second. The
first 25 seconds mimics the dissociated state of a Cy3–Cy5 pair. (C)
Histograms of the apparent EFRET (= IA/(IA + ID); ID and IA are the
fluorescence intensities of the donor and acceptor, respectively) for
nanovesicles containing a single Cy3 (line patterned columns) and for
nanovesicles containing a free Cy3 and Cy5 molecule (clear columns).

The dissociated state can be mimicked by a nanovesicle containing a free

donor and a free acceptor (Fig. 4.3B), as the free dyes do not interact with
each other. Here, the existence of both a donor and an acceptor must be
confirmed by separate laser excitations (Fig. 4.3B). Under 532-nm
excitation, the apparent EFRET is 0.15 ± 0.14 (Fig. 4.3C); the larger value here
compared with that of Cy5-blinked state is likely due to the residual direct
excitation of Cy5 fluorescence by the 532-nm laser and some energy
transfer of Cy3–Cy5 due to their confined coexistence inside the
nanovesicle.

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Riboswitch Discovery, Structure and Function

Bassem Shebl, ... Peter V. Cornish, in Methods in Enzymology, 2014

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4.2 Choosing a dye
An ideal fluorescent dye does exhibit a few favorable properties. First, it is
photostable, thus resisting photobleaching at least at the timescale of the
dynamic motion under investigation. Second, a dye with minimal intensity
fluctuations is required. In addition, one picks bright dyes, i.e., choosing the
highest possible extinction coefficient and quantum yield. Equally
important, the excitation and emission ranges of the dye lie within the
attainable visible region of the spectrum. Also, the dye is small enough not
to perturb the structural integrity of the investigated biocomplex nor affect
its activity. Finally, the dye needs to be available in a chemically usable form
allowing specific attachment to the molecule of interest. Few dyes provide
good examples of the aforementioned properties: Alexa, Atto, cyanine, and
tetramethylrhodamine (TMR). Alexa and Atto have been used for diffusing
molecules (Munro, Altman, Tung, Sanbonmatsu, & Blanchard, 2010 ).
Cyanine dyes and TMR have been used for immobilized molecules.

However, smFRET deals with donor–acceptor dye pairs rather than a single
dye. Correspondingly, an ideal FRET dye-pair has compatible features. For
example, the quantum yields of both dyes are comparable to one another
to facilitate data analysis. Furthermore, the dyes show a good spectral
overlap between the donor dye emission and the acceptor dye absorption.
At the same time, a large separation between the emission spectra of the
dyes is desirable to minimize crosstalk between donor and acceptor dye
channels on the detector. With that in mind, Cy3 and Cy5 are the most
commonly used FRET pair since they have good spectral separation and
comparable quantum yields (Joo & Ha, 2008). In addition, Cy3 and Cy5 are
commercially available with a wide range of derivatives, such as: NHS-
esters, maleimides, and azides (Lumiprobe Life Science Solutions and GE
Healthcare). Figure 16.4 and Table 16.1 show several labeling schemes
used in studying ribosome dynamics, which can be extrapolated to
comparable biological platforms.

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Electrokinetics in Microfluidics
In Interface Science and Technology, 2004

Chemicals
Two caged fluorescent dyes, supplied by Molecular Probes, were employed
here: 5-carboxymethoxy-2-nitrobenzyl (CMNB)-caged fluorescein (826.81
MW); and 4,5-dimethoxy-2-nitrobenzyl (DMNB)-caged fluorescein dextran
(10000 MW). Both dyes were dissolved in sodium carbonate buffer of pH =
9.0. The buffer was prepared by dissolving 39.8×10-3 mol of NaHCO 3 and
3.41×10-3 mol of Na 2CO3 in 1 litre of pure water resulting in a solution of
ionic strength, I = 0.05. Stock solutions of DMNB-caged fluorescein dextran
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and CMNB-caged fluorescein were prepared in 0.2 mM and 1mM
concentrations respectively. The solutions were aliquoted and stored in
darkness at -20°C. Immediately before use, all solutions were filtered using
0.2μm pore size syringe filters.

Both caged dyes release fluorescein upon photolysis which has an

absorption maximum at approximately λ = 490nm and is well suited to
excitation with the λ = 488nm output line of the argon laser. DMNB caging
groups absorb light most efficiently from λ = 340nm to λ = 360nm. CMNB
caging groups, however, exhibit an absorption maximum at λ = 334nm
which is very well suited to the λ = 337nm output of a nitrogen laser.

To facilitate the multiple-image analysis technique, all results presented

here were acquired using mode 2 in which the camera ran in progressive
scan video mode and the delay generator controlled the ultraviolet laser.
The nitrogen laser was triple-pulsed at 30Hz at each uncaging event. The
overall run frequency was set to 0.15 Hz, providing more than sufficient
time for uncaged dye to exit the field of view. The camera was run at 15 Hz
resulting in 100 stored images per uncaging event. Individual exposure
times were 1/125 sec (corresponding to shutter mode 8 on the TM-9701).
The timescale over which the uncaging process occurs is also an important
consideration. In general, this delay can vary from microseconds to
seconds [21]. Here, dye release was effectively complete 50msec after the
uncaging event, and only images taken after this period were used in
velocimetry calculations. This delay corresponds to the first major
timescale of dye release found by Lempert et al. [6].

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Nanomedicine in Theranostics
Renu Geetha Bai, ... Sivakumar Manickam, in Nanotechnology Applications for
Tissue Engineering, 2015

12.4.1 Role of QDs in Bioimaging

The inorganic fluorescent dyes made of semiconductor nanocrystals with
unique optical and chemical features are referred as QDs. They are highly
stable and resistant to photobleaching compared to the normal organic
fluorophores. QDs are made of atoms from groups II–VI (e.g., CdSe, CdTe,
CdS, and ZnSe) or III–V (InP and InAs) elements in the periodic table. By
varying the crystal size (usually <10 nm), the emission spectrum of QDs can
be changed and it is possible to excite different-sized nanocrystals with the
same wavelength light or a single light source. QDs are nanomaterials with
physical dimension less than exciton Bohr radius and due to the quantum
confinement they have specific optical and electrical properties. These
properties enable them to function as ultimate fluorophores for
ultrasensitive detection, multitarget, and multicolor fluorescence assays
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[16,17]. The interactions between electrons, holes, and their local
environments determine the optical properties of the QDs. When the
excitation energy of the semiconductor exceeds the bandgap between the
valance and conduction band, the QDs absorb energy in the form of
photons and the electrons will be transferred from valence band to the
conduction band. UV–visible spectrum analysis gives the detailed
information about the excitation emission spectrum of the QDs with the
same and different wavelengths [18]. In order to maintain the quantum
yield and the stability in aqueous solutions, QDs need to have a surface
covering, where the surface coating maintains the stability of the
fluorescence. For in vivo applications, QDs should have a long circulating
time, less degree of nonspecific accumulation, and long-term stability of
fluorescence. The surface modification agents used are polymers, such as
poly(acrylic acid) and polyethylene glycol (PEG). The in vivo imaging of the
surface-modified QDs with the enhanced properties was analyzed using
different microscopic imaging tools and extended fluorescence stability up
to 4 months was reported [17]. Generally, a typical QD is comprised of
three parts: a core, a shell, and a coating. The core is composed of
thousands of semiconductor atoms; shell is to stabilize the core; and
coatings of polymers are to make the QD hydrophilic for ease of
conjugation with ligands. Phospholipid-encapsulated biocompatible QDs
were used for the in vivo imaging of human prostate cancer cells induced
on mice. The core–shell, CdSe–ZnS is targeted by a ligand (prostate-specific
membrane antigen monoclonal antibody) and covered by an amphiphilic
polymer, tri-n-octylphosphine oxide (TOPO) coating. These probes were
efficient to bind to the specific receptors and were bright, stable as well as
suited for conjugation and were employed for imaging or diagnosis
purposes [19].

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