Journal of Fluorescence

https://doi.org/10.1007/s10895-021-02770-9

REVIEW

Fluorescein Based Fluorescence Sensors for the Selective Sensing

of Various Analytes
Keerthana S1 · Bincy Sam1 · Louis George1 · Sudhakar Y. N1 · Anitha Varghese1

Received: 25 April 2021 / Accepted: 30 June 2021

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract
Fluorescein molecules are extensively used to develop fluorescent probes for various analytes due to their excellent pho-
tophysical properties and the spirocyclic structure. The main structural modification of fluorescein occurs at the carboxyl
group where different groups can be easily introduced to produce the spirolactam structure which is non-fluorescent. The
spirolactam ring opening accounts for the fluorescence and the dual sensing of analytes using fluorescent sensors is still a
topic of high interest. There is an increase in the number of dual sensors developed in the past five years and quite a good
number of fluorescein derivatives were also reported based on reversible mechanisms. This review analyses environmentally
and biologically important cations such as C ­ u2+, ­Hg2+, ­Fe3+, ­Pd2+, ­Zn2+, ­Cd2+, and M
­ g2+; anions (­ F−, ­OCl−) and small
molecules (thiols, CO and ­H2S). Structural modifications, binding mechanisms, different strategies and a comparative study
for selected cations, anions and molecules are outlined in the article.

Keywords Fluorescein · Fluorescent sensors · Spirolactum · Fluorescent probes

Introduction A large variety of fluorophores like coumarin [7], xan-

Detection and quantification of biologically and environ-
mentally important species is of great interest in recent
times. Several methods including gas chromatography [1],
liquid chromatography-mass spectrometry [2], conductom-
etry, electrochemical analysis [3], colorimetry, and spectro-
photometry can be used for the sensing of analytes [4]. But
among these, fluorescent sensing can be considered as most
effective due its high selectivity and sensitivity, low detec-
tion limit and simplicity in handling the sensors [5]. Also
fluorescent sensors have an advantage over other techniques
that they can be applied on living cells for systematic testing
with real time monitoring. A fluorescent probe consists of
fluorophore, linker and a recognition site. The sensor reacts
with the analyte either through chemical modification fol-
lowed by binding to the target or through enzyme catalysis
[6].
* Anitha Varghese
anitha.varghese@christuniversity.in
Louis George
louis.george@christuniversity.in
1
Department of Chemistry, CHRIST (Deemed To Be
University), Hosur Road, Bengaluru 560029, India
thenes [8], BODIPY [9], pthalimide [10] and quinolones
[11] are available as chemosensors with unique properties.
Among all these, xanthene dyes (Fig. 1) are unique due to their
excellent photostability, high quantum yield, high absorp-
tion coefficient and long emission wavelength. Xanthene
contains two benzene rings connected through an oxygen
atom and methylene group. Since the xanthene unit is a part
of the fluorescein molecule, it belongs to the xanthene class
of dyes and shows excellent fluorescent properties [12]. This
review discusses fluorescein derivatives developed in the last
ten years for the detection of metal ions, anions and small
molecules.
Fluorescein Derivatives As Fluorescent
Probes
Fluorescein is a type of xanthene dye with yellowish green
fluorescence which was first synthesized by von Bayer in
1871 using resorcinol and phthalic anhydride via Friedel
craft acylation/cyclodegradation reaction [13]. It has a rigid-
coplanar tricyclic structure with two aryl groups fused into a
pyran ring. The fluorescein family of dyes have two distinc-
tive structures, a fluorescent ring opened carboxylic acid form

13
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and a non-fluorescent ring closed spirocyclic lactone form.
The spirocyclic structure is colorless and non-fluorescent,
but the importance of fluorescein structure lies in the
spirolactam structure. This is due to its characteristic ‘open
and close’ response to specific environments. The spirolac-
tam structure of fluorescein can open up the ring in addition
to specific analytes emitting bright signals with high molar
absorptivity and excellent quantum yield [14, 15] thereby
induce color changes and fluorescence enhancement. The
open-close equilibrium structure of fluorescein makes it sen-
sitive to pH of the medium [16]. This pH sensitivity of fluo-
rescein can be used to sense changes in a specific environ-
ment by monitoring its ring opening and closing equilibrium
in addition to other spectroscopic effects to detect metal ions
from industrial and commercial samples [17].
The fluorescein structure can undergo modification in
two parts, the xanthene ring and benzene moiety which
lie orthogonal to each other. In majority of the derivatives
modification occurs at 3, 4, 5, 6, 2’, 4’, 5’ positions or by
modifying the hydroxyl group or replacing the oxygen atom
at 10 positions (Fig. 2) [18].
Different sites are employed for structural modification
and linker attachment in fluorescein molecule. The sites for
attachment are often modified with substituents to improve
the fluorescence properties of dye. Different types of rea-
gents in the synthesis of fluorescein moieties involve con-
densation of aldehydes, carboxylic anhydrides and phenol
derivatives [19].
The carboxyl group present on the benzene moiety can be
modified using Schiff’s base reaction which leads to the for-
mation of fluorescein hydrazine that connects with the alde-
hyde containing groups and these are used as binding sites
for some of the metal ions [20]. High activity of the Schiff’s
base provides more binding sites improving the sensitivity
of detection. The xanthene ring can be modified to obtain
fluorescein aldehyde through Reimer-Tiemann reaction
forming amino group containing probes having lone pair
of electrons which through photo-induced electron transfer
(PET) process leads to fluorescence quenching. This can
be solved when it combines with metal ions having empty
orbital leading to fluorescence recovery. Fluorescein probes
are commonly used to detect metal ions such as copper, mer-
cury, iron, palladium, cadmium, and magnesium in aqueous
solutions and living cells which are of great significance for
human health.
Initially the probe is colorless and non-fluorescent due to
the breaking of π bonds. Addition of ions leads to ester bond
breaking and small organic molecules removal resulting in
notable color and fluorescence change. The benzene moiety
is modified connecting the fluorescein structure with organic
fluorophores or nanoparticles like gold [20].
The connection of fluorescein with organic fluoro-
phore is used to detect analytes based on the fluorescence
13
Journal of Fluorescence
resonance energy transfer (FRET) process. This is a ratio-
metric fluorescein detection of metal ions. Fluorescein
connected with AuNPs induces fluorescence quenching
through the FRET process. But some of the analytes can
hinder the FRET process due to strong binding of the ana-
lyte leading to fluorescence recovery. The site containing
hydroxyl group at ortho position have been modified with
halogen to improve the fluorescence properties of the dye
[20]. This molecular orientation of the dye plays a key role
in many potential applications such as in colorimetric sen-
sors, [21] fluorescent sensors [22], photographic industry,
[23] and molecular optoelectronic devices [24].
Structure of fluorescein provides possibilities in design-
ing sensors for a wide range of analytes (Fig. 3). It has
been derivatized into versatile fluorescent reagents com-
prising different reactive functional groups. Fluorescein
exhibits good water solubility, visible excitation and emis-
sion (an absorption maximum at 494 nm and emission
maximum of 521 nm in water) showing maximum bright-
ness at physiological pH. Very high molar absorptivity,
large fluorescence quantum yield, high photostability, long
absorption and emission wavelength and large extinction
coefficient makes it useful and sensitive as a fluorescent
label. Based on these photophysical properties of fluores-
cein, these dyes are usually employed to construct optical
sensors for many target metal ions. Therefore, these dyes
have become one of the most significant classes of fluo-
rescent probes. Fluorescein itself does not have a coupling
group and hence, many researches has been focused on
its derivatives with different functional groups attached at
its different positions such as, 5(6)-carboxy-, amino-, and
isothiocyanato fluorescein [14] which is discussed in this
review. Fluorescein and its derivatives are considered to be
excellent chromophores due to their good water solubility,
high photostability and short synthetic routes [25]. They
have been used as a powerful tool for molecular studies in
biology, interactions between ligands and the molecules,
drug discovery and in environmental research.
Some of the common fluorescein derivatives include
carboxyfluorescein in which the –CO2H group is attached
to 5th or 6th position of the fluorescein molecule. These
–CO 2H groups can be further activated using different
functional groups. Carboxyfluorescein generated can
be further halogenated to produce a better derivative.
5(6)-Hydroxymethylfluorescein and 5(6)- aminomethyl
fluorescein can be synthesized by replacing one of the
hydrogen from methyl group substituted at 5th or 6th
position by –OH and –NH2 groups. By substituting –NH2
group at 5th or 6th position, aminofluorescein can be pre-
pared. These derivatives are highly useful for structural
elucidation of proteins and membranes, lipid transport and
metabolism because the –NH2 group can couple with the
carboxylic group in target molecules. Azidofluorescein can
Journal of Fluorescence
be synthesized by attaching an azide group to 5th or 6th
position of fluorescein.
Fluorescein Based Fluorescent Probes
for Detection of Cations
Fluorescent sensors are known to extensively sense metal
ions through fluorescence signal changes [26] and are gain-
ing much attention due to their high sensitivity, selectiv-
ity and ease of measurement [27–31]. These probes or
chemo sensors can change color or fluorescence intensity
upon interacting with selective ions [32, 33]. Recognition
and sensing of biologically and environmentally important
metal ions is a significant accomplishment in the field of
chemosensors [34–42]. Fluorescent chemosensors consists
of three components- receptor (ionophore), fluorophore (sig-
nalling moiety), and a chelator.
Detection of Copper Ions
Cu2+ is the third most abundant metal ion in the human
body which is needed for proper growth and development
of the body [43]. It plays important roles in different met-
alloenzymes, human nervous system and gene expression,
protein functioning and biological systems of plants. Also
copper compounds are involved in plant disease treatment,
water treatment and preservatives for wood and leather [44].
Deficiency of these can induce effects on human health and
lead to diseases like anaemia and arteriosclerosis. But excess
intake of these compounds can lead to serious neuro-gener-
ative disorders ranging from amyotrophic lateral sclerosis,
Parkinson’s, Alzheimer’s, Wilson’s and Menke’s diseases
[45]. There can also be the possibility of kidney damage due
to excess intake of this ion [46]. Hence, detection of ­Cu2+
and its compounds are essential. Several fluorescent probes
for ­Cu2+ ions have been reported in literature including
rhodamine based, BODIPY based, coumarin based, anthra-
cene based [47] and fluorescein-based probes. Among these
rhodamine sensors have some deficiencies like small stokes
shift which limits its application in biological systems which
turned the attention to fluorescein-based probes. Diwan and
co-workers revealed that fluorescein derivatives are able to
give turn on response for various metal ions, especially ­Cu2+
in visible to NIR region [48]. They synthesized a fluores-
cein-sugar conjugated chromo- fluorogenic (FG) probe 1.
The inclusion of galactose into FG led to its high selectivity
and sensitivity towards copper ions and also showed good
water compatibility.
The major disadvantages of these types of chemosensors
are that they absorb in ultraviolet and visible regions and
show changes in fluorescence in the same region. Hence,
it is necessary to develop NIR absorbing and emitting
chemosensors. Silica provides a hydrophilic surface due
to presence of silanol groups which are weakly acidic and
also provide sites for chemical modification [49, 50]. Fluo-
rescein hydrazide modified silica nano-particles FH-SNPs
(probe 2) has been reported for the detection of copper ions.
The selectivity of the probe towards C ­ u2+ was observed to
be due to FH opening its structure upon reacting with cop-
per ions [51]. Xiaofeng Bao and co-workers synthesized
fluorescein derivative (probe 3) E)-2-(((1H-pyrrol-2-yl)
methylene)amino)-3’,6’-dihydroxyspiro[isoindoline-1,9’-
xanthen]-3-one (FLPY). Mechanism suggest that the strong
fluorescence was observed due to the hydrolytic cleavage of
N = C induced by the copper ions resulting in a ring opened
fluorescein hydrazine [52].
1-phenyl-3-methyl-5-hydroxypyrazole-
4-benzoyl(fluorescein) hydrazine was considered to be a
fluorescein-based colorimetric sensor (probe 4), for rapid,
selective and sensitive response to ­Cu2+ in aqueous medium.
The significance of this molecule lies in the chelating ability
towards copper ions, which on coordination the spirolactam
of fluorescein moiety can be opened [53].
Most of the reported ­Cu2+ detection using colorimetric
chemosensors possess many disadvantages such as poor
detection limit, less response time, and interference of other
metals. Taha M. Elmorsi and co-workers developed a new
fluorescein based colorimetric sensor (probe 5) benzo[f]fluo-
rescein derivative (BFFNH). The compound was found to
show sensitivity at alkaline condition [54]. Although many
reported literature shows the selectivity of probes in aque-
ous and organic media, ligands are rarely found active in
both these media. Imine-functionalized fluorescein based
ligand (probe 6) was found to be binding selectively to
copper ions without affecting selectivity of other cations
in both aqueous and organic medium. Fluorescein based
Schiff base ligand was synthesized which coordinates with
lewis acid metal ions (Fig. 4) [55]. 5-chlorosalicylaldehyde
fluorescein hydrazine (CSFH) (probe 7) consists of carbonyl
O, amido N and hydroxyl O atoms which act as donors
for binding to copper ions [56]. A novel fluorescein sen-
sor compound based on furfuraldehyde fluorescein hydra-
zone (FFH) (probe 8) was synthesised by the reaction of the
amino group of fluorescein with furfuraldehyde forming a
Schiff base which can coordinate with oxygen atom which
opens up the fluorescein moiety and the spectra change
can be recorded [57]. The spiro form fluorescein hydrazide
(probe 9) bears amide bond which get cleaved when bound
with ­Cu2+ causing release of fluorescein producing fluores-
cence [58]. Most of these probes are analysed separately
and isomers of the probes are never being studied. While
Guanhong Liu and his co-workers synthesized probe 10
using two isomeric fluorescein hydrazone derivatives such
as 2-Hydroxy-5-methoxybenzaldehyde (5-HMBA-FH) and
2-Hydroxy-3-methoxybenzaldehyde (3-HMBA-FH) to
13

detect ­Cu2+ ions. The two isomeric probes can readily react
with fluorescein hydrazide, and these two contain nitrogen
atom which could easily combine with copper ions [59].
Some turn-on fluorescent probes are highly sensitive and
variation in the concentration and environment of the sample
can cause change in the accuracy of fluorescent measure-
ments. Hence, to avoid this, ratiometric fluorescent probes
are designed. FRET is observed to be one of most efficient
sensing mechanisms for ratiometric probes where excited
donor fluorophores transfer its energy to acceptor fluoro-
phores [60]. Mondal et al. devised a FRET off–on sensor
(probe 11) using phenanthroline donor and fluorescein for
selective detection of ­Cu2+ as no fluorescence emission was
observed in the absence of copper ions. In addition, the
spirocyclic ring opens showing green fluorescence where
copper binds through two nitrogen atoms of phenanthroline
and two enamine N of fluorescein moiety which is effec-
tively applied for the detection of copper ions in aqueous
environment and in living cells [61]. Fluorescein isothio-
cyanate functionalized gold nanoparticles (FITC-AuNPs)
(probe 12) was reported as a highly sensitive probe. There
are unsaturated gold atoms present on the surface of AuNPs
with presence of unoccupied orbitals, while the isocyanate
group of FITC molecule known as strong nucleophile dis-
place citrate ions and form stable complex with gold in sur-
face of AuNP [62]. Prasad G. Mahajan and his co-workers
synthesized small fluorescein based sensor (probe 13) named
N-(3’,6’-dihydroxy-3-oxo-3,3a-dihydrospiro[isoindole-1,9′-
xanthene]-2(7aH)-yl)-1-naphthamide (FNH). These fluores-
cent based organic nanoparticles have been adopted as they
could overcome the drawbacks of the traditional analytical
methods. These nanoparticles are advantageous in terms
of high selectivity, sensitive response, ease of preparation
and use in aqueous medium directly [63]. Imidazole and
its functionalised derivatives have been applied in many
bioactivities. Helal et al. synthesized (probe 14) fluorescein
hydrazone and N-methylimidazole for detection of ­Cu2+
ions. Introduction of methyl group into the imidazole moiety
increases electron donating capability of imidazole which
makes it more sensitive towards copper ions [64]. A new
using photochromic diarylethene with a fluorescein via a
triazole linkage (probe 15) was reported and found that its
fluorescence intensity can be increased by addition of C ­ u2+
2+
ions [65]. A new spectrophotometric method for ­Cu ions
using 2′, 7′-di(2-benzothiazolyl)-fluorescein (BTF) probe 16
was synthesized based on cloud point extraction(CPE) and
the method has been successfully used for determination of
copper in tea and water samples [66].
The color change of the chemosensor was important for
monitoring ions through unaided eye as no complicated
instruments were required. The colorimetric chemosensors
proved to be highly selective towards copper ions. Probes 4
and 5 are synthesized using colorimetric based fluorescein
13
Journal of Fluorescence
chemo sensors, but usage of these colorimetric probes is
limited due to the low detection limit and interference from
other ions. Probe 7 is a chlorine introduced salicylaldehyde
fluorescein hydrazone. Aldehyde is a reactive group and is
capable of forming Schiff base by reaction with amino group
which allows the copper ions to bind through carbonyl car-
bon, amido N and hydroxyl O as donors. Hence, aldehyde
introduced probes can serve as effective sensors in future
research directions. The probes composed by AUNPs also
showed excellent sensitivity and selectivity over other tra-
ditional fluorescein moieties. The water compatibility of the
probe was also improved by the introduction of sugar moiety
into fluorescein as it shows a plausible solvent effect to bind
the metal ions.
Detection of Mercury Ions
Mercury is considered as one of the most toxic heavy met-
als [67] as it can cause severe health problems by damag-
ing the central nervous system, brain, kidney and endocrine
system [68]. Even millimolar levels of mercury are toxic
and therefore it is very important to monitor its concentra-
tion in biological systems. Several fluorescent sensors have
been developed for detection of ­Hg2+ [69, 70]. Some of the
fluorescein derivatives used as sensors are discussed further.
Many chemosensors were developed using dichlorofluo-
rescein (DCF) as the fluorophore [71]. A probe developed
from dichlorofluorescein using coumarin moieties behaves
as the internal standard for signalling of mercury ions (probe
17). For different metal ions the responses at DCF moiety
are different but the usage of coumarin as internal standard
was more convenient for the ion analysis. Complexation of
­Hg2+ with two adjacent piparazine moieties explained the
selective sensing [72]. Chemosensors based on the modifica-
tion of hydroxyl and carbonyl group of fluorescein were well
known but only few studies were done on the metal coor-
dination at the carbonyl group of xanthene moiety [73]. To
study the metal coordination effect, two probes 18a and 18b
were developed by Yi-Bin Ruan et al. First one was a meth-
ylated fluorescein and second one was developed by replac-
ing these methyl groups with two triazolyl amino esters [74].
One reason known for the high toxicity of H ­ g2+ is its high
affinity towards the thiols. Yusha Feng et al. made use of this
property of H­ g2+ and developed probe 19 by introducing a
thiol group onto fluorescein. The probe combines with ­Hg2+
through imino N and thioether S atoms of both ligands and the
ring opened amide form caused the fluorescence enhancement
(Fig. 5) [75]. Solubility of the probes in pure water was still a
challenge faced while developing new sensors. Shanyi Guang
et al. addressed this issue and developed a new probe 20.
Water solubility was improved by introducing a non-hydrogen
bonding group which would reduce the intermolecular hydro-
gen bonding. Fluorescent enhancement was observed when
Journal of Fluorescence
oxygen and nitrogen atoms of the probe combined with ­Hg2+
to form the stable five membered rings [76].
A new probe developed by incorporating different sul-
fur functional groups into fluorescein by using the PET
mechanism exhibited good fluorescence and high membrane
permeability [77]. Development of reusable sensors was a
major challenge for researchers. Zixiang Qu et al. synthe-
sized sensitive polymers bearing fluorescein molecules as
sensors (probe 22) which were reusable. N-(fluorescein)
lactam-N′-methylethylidene (FLA) is developed first and
the hydrogel sensor is developed using free radical polym-
erization. Acrylamide (AAm), 2-hydroxyethyl methacrylate
(HEMA) and methyl methacrylate (MMA) were treated with
FLA to obtain the hydrogels. Fluorescence enhancement was
observed on adding H ­ g2+ which break the spirolactam ring
and block PET process [78].
One challenge faced by H ­ g2+ sensors was fluorescence
quenching due to the spin orbit interactions. The approach
used for developing each sensor was different. Fluorescence
spectra studies of the sensors showed that on adding mercury
ion a 30, 13, 37, and 51- fold increase was observed for probe
17, 18, 19 and 21 respectively. A large stokes shift value
of 67 nm for probe 20 conveyed that the complex formed
with the mercury ion was quite stable. Sensor 18 consid-
ered to examine the effect of substituent (triazolyl group)
on the xanthene ring and noticed that there was no change
in the photophysical properties. In addition, the group in the
sensor is just a linker and no change was observed in the
binding mechanism between the carbonyl and mercury ion.
Applicability studies showed that probe 22 can be applied in
manufacturing devices which can also be used as unaided eye
sensors in successfully detecting mercury ion.
Detection of Iron Ions
Iron is one of the most important transition trace metals
of all living cells [79]. Among metal ions, iron performs a
crucial role in a wide range of biological processes, while
several biological functions depend on the concentration
of iron to maintain homeostatic mechanism of biologi-
cal systems [80]. Its deficiency causes numerous health
problems such as iron deficiency anemia, sickle cell ane-
mia, thalassemia and hemochromatosis. A wide range of
methods have been investigated for the detection of iron,
such as inductively coupled plasma (ICP), HCLP, atomic
absorption spectroscopy, electrospray ionisation (ESI) and
fluorescence-based methods [81]. Yan Gao et al. synthe-
sized a novel colorimetric and fluorescent chemosensor
(probe23) (E)-2- ((benzo[d]thiazol-2-ylmethylene)amino)-
3’,6’-dihydroxspiro[isoindoline-1,9’-xanthen]-3-one(1)
based on fluorescein fluorophore for the quantification of
­Fe3+ ions [82]. The probe binds with ­Fe3+ ions through
nitrogen atoms of CH = N of the chemosensor. Catechol
derivatives serve as important molecules due to their
intrinsic acid base properties. A fluorescein based probe
containing a catechol chelating unit for the detection of
­Fe3+ ions (probe 24) was prepared by ortho substitution
of methyl amine in 1,2-dihydroxybenzene. No reports on
detection of F ­ e3+ ions using sensors like PAF have been
reported earlier. PAF have been considered as novel func-
tional materials because their rigid aromatic components
can be easily functionalized without any collapse of skel-
eton and also due to their high thermal stability [83]. A
fluorescein probe based on porous aromatic framework
(PAF) through fluorescence quenching mechanism exists
in the literature [84]. The color of the probe appears only
on addition of 5-carboxyfluorescein (5-CF) into PAF
(probe 25) and found to be more selective and sensitive to
iron than PAF alone (Fig. 6).
Studies on the detection of metals using graphene struc-
ture are rare. Ays Merve S et al. synthesized fluorescence
sensor (probe 26) using fluorescein reduced graphene oxide
with polyethyleneimine (PEI) [85]. Among graphene, gra-
phene oxide (GO) and reduced graphene oxide (r-GO), r-GO
have received much greater attention. GO contains many
oxygen containing groups and these are insulators, while
eliminating an oxygen containing functional group makes
it a conductor and also shows good mechanical properties.
r-GO is modified using PEI makes it water soluble and
amino groups can bind to ­Fe3+ ions for their easy detection.
Graphite is of course a cheaper alternative, but it possesses
relatively low surface area.
The combination of catechol derivative and different
functional group moieties in probe 24 is very appealing for
the sensitive and selective detection of iron. The introduc-
tion of different functional groups into the covalently cou-
pled catechol unit having aromatic ring provides a sensitive
receptor site for iron to bind. The absorption value of the
probe as indicated in Table 1 differs greatly in comparison to
other sensors. This is observed because of the degradation of
the compounds attached to the catechol fragment indicating
their limit in application. The poor thermal stability and acid
resistance of MOF (metallic organic framework) and COF
(covalent organic framework) is overcome by use of porous
aromatic framework as in probe 25. PAF is composed of
phenyl ring derived fragments which can be easily modi-
fied with different functional groups. This provides a large
surface area and has a large amount of free space to accom-
modate guest molecules facilitating condensation of active
species which lack in probe 26 as graphite provides a low
surface area. The open pores in PAF are advantageous for
incorporating PAF with complementary functional groups
to obtain advanced activities that can serve as a better option
for the selective detection of iron.
13
 Journal of Fluorescence

Table 1  Comparison of different sensors used for the detection of cations

Analyte Probe Solvent 𝜆 ex Binding Detection Reference
/Association Limit
Constant (M)
(M−1)

Cu2+ 1 HEPES buffered solution (­ H2O:CH3CN = 70:30, 515 nm (1.02 ± 0.08) × 105 6.32 × 10 − 9 [48]
pH = 7.4)
2 Tris HCl buffer, pH = 5.2 518 nm - 1.8 × ­10–2 [51]
3 DMSO/HEPES pH 7.2 504 nm 1.29 × ­104 0.296 [52]
4 DMSO/ 495 nm 3.3 × ­104 [53]
H2O
5 Tris–HCl buffer pH = 7.2 455 nm 3.16 × ­104 0.50 M [54]
6 acetonitrile 496 nm, 2.23 × ­104 20.0 × ­10–6 M [55]
7 EtOH 495 nm 6.85 × ­105 0.25 × ­10–6 [56]
8 EtOH 502 nm 6.1 ×104 mol/L [57]
9 Tris–HCl buffer (pH 7.2) 495 nm 64 × ­10–9 [58]
4
10 acetonitrile 498 nm 2.54 × ­10 mol/L 0.442 × ­10–6 and 3.682 × ­10–6 [59]
and
3.58 × ­104 mol/L,
11 HEPES, pH = 7.4, methanol 506 nm - - [61]
12 Na2HPO4–NaH2PO4 buffer pH = 6.8 520 nm 0.37 - [62]
13 Acetone 365 nm 2.564 × ­104 0.024 × ­10–6 [63]
14 HEPES buffer at pH 7.4 502 nm 3.09 × ­104 0.037 × ­10–6 [64]
15 tris-buffer pH 7.4 435 nm - 10 × ­10–9 [65]
16 Methanol 559 nm - 7.48 [66]
Hg2+ 17 DMSO solution, pH 4.8 340 nm 4.3 ­10–6 mol ­L−1 [72]
18 THF 445 nm 39 nM [74]
19 EtOH/HEPES buffer (1:1, v/v, pH 7.4) 509 nm 3.90 × ­10 − 8 M [75]
20 Aqueous solution 445 nm 0.86 × 10 − 9 mol·L − 1 [76]
21 HEPES buffer, pH = 7.4 470 nm 1.10 × ­10−7 mol·L−1 [77]
22 DMSO and ­H2O (5/1, v/v) 490 nm 14.9 nM [78]
C2H5OH/H2O (5/1, v/v) 435 nm
Fe2+ 23 DMSO/H2O HEPES at pH 7.2 393 nm 1.085 × ­105 7.4 × ­10–9 [82]
24 Methanol 493 nm 4.08 - [83]
25 Ethanol 365 nm - 3.8 × ­10–5 [84]
26 Ethanol 456 nm - 1.12 [85]
Pd2+ 27 DMF 520 nm 0.03 × ­10–6 [91]
28 MeOH - 0.06 × ­1012 [92]
29 NaOH 511 nm 1.07 × ­10–6 [93]
30 Ethanol 492 nm - [94]
Zn2+ 31 1:1 ethanol-HEPES buffer (v/v, pH 7.2) 400 nm pka-9.95 - [99]
32 water/ethanol, (1:9, v/v; 10 mM HEPES; pH 404 nm 1.213 × ­104 ­mol−1 6.54 ppb [100]
=7.4)
33 CH3CN–PBS (10 mM, pH 7.4) 490 nm 2.4 × 104 M ­ −1 1.2 ×10–6 M [101]
34 HEPES buffer (pH = 7.4, 10 mM) 365 nm 9.28 × ­10 ­M−1
6
5.49 × ­10–8 M [103]
35 ethanol–water (9:1, v/v, 15 μM HEPES buffer, 410 nm 2.86 × ­104 ­M−1 1.59 μM [105]
pH 7.2)
36 MeCN-H2O (v/v, 2:1) 364 nm 3.58 × ­104 ­M− 1 5.64 μM [106]
2+
Cd 37 PBS 470 nm 12.08 nM [113]
38 20 mM HEPES buffer at pH 7.4 470 nm - [114]
39 THF (2.0 × ­10–5 mol ­L−1) 350 nm 6.5 × ­10–7 mol ­L−1 [116]
Mg2+ 40 Ethanol 489 nm - [122]
41 ethanol 323 nm 2.67 × ­10–8 ­M−1 [124]

13
Journal of Fluorescence

Table 2  Comparison of results of different samples used in the detection of two cations
NO OF CATIONS PROBE SOLVENT ± Detection limit ­M−1 Color change Reference

Cu2+ and ­Al3+ 42 EtOH-H2O 557 nm 7.32 × ­10–8 for ­Al3+ and 1.47 × ­10–8 for ­Cu2+ Yellow [125]
Al3+ and ­Zn2+ 43 EtOH 384 nm 5.37 × ­10−8 for ­Al3+ and 7.90 × ­10−8 for ­Zn2+ blue [126]
Cd2+ and ­Co2+ 44 EtOH-H2O 456 nm - - [127]
Hg2+ and ­Zn2+ 45 EtOH-H2O 521 nm 0.54 µM for Z ­ n2+ and 1.16 µM for ­Hg2+ yellow [128]

Detection of Palladium Ions
Pd catalysts are employed in carbon–carbon bond formation
and play an important role in synthesis of complex struc-
tures such as natural products, pharmaceuticals, polymers
and super molecules [86, 87]. In the healthcare and pharma
sector, Pd is used in dental alloys and surgical instruments.
The widespread use of Pd causes contamination in drugs and
consequential health risks. Pd could destroy DNA, coordi-
nate with thiol containing amino acids and trigger protein
denaturation causing cellular damages [88–90]. Hence, real
time monitoring and detection of these ions are important.
Mithun Santra et al. synthesized fluorescein based probe 27
propargyl ether to detect palladium ions at room tempera-
ture. The propargyl ethers are cleaved by palladium using
depropargylation reaction and fluorescein could sense palla-
dium in three different oxidation states. The probe also could
detect fluorescently the palladium ions in zebrafish [91].
To overcome the issue of background fluorescence,
Weston R. Kitley et al. synthesized an open-closed equi-
librium of fluorescein probe 28 (bis allyl ether) to achieve
greater contrast and sensitivity to palladium. This proves to
be an attractive method as the probe could sense palladium
by introduction of allyl groups into phenolic fluorophores
and through deallylation these groups are removed and
releases fluorescent molecule with an increase in fluores-
cence [92]. Carbon nanoparticles are used as alternative
probes compared to their carbon substrates due to their
lower cost, widespread availability and very high surface
area to volume ratios. Hence, functionalized carbon nano-
materials which exhibit visible fluorescence emission were
prepared by coupling the carbon nanomaterials with fluo-
rescein. Janjira Panchompoo et al. synthesized fluorescein
modified carbon black nanomaterials (CB) (probe 29) for
effective detection of palladium(II). The fluorescence emis-
sion spectra were studied by suspending the carbon black
nanomaterials in water and then bubbled with ­N2 gas to get
rid of oxygen since presence of oxygen quench the fluores-
cence [93]. Guo Wei et al. synthesized novel fluorescein
based derivative chemosensor (probe 30) for the detection
of ­Pd2+ [94]. The fluorescein is treated with hydrazinium
hydroxide in EtOH. Treatment with various metals, the
sensor emits very weak fluorescence due to protected
hydroxyl groups by alkyl groups. Only in the presence of
palladium the protected hydroxyl group of sensor hydrolyse
to produce fluorescein leading to fluorescence change.
Probe 27 is proven to be an effective sensor for palladium
detection as this involves no addition of reagents to sense

Table 3  Comparison of results Analyte Probe Solvent 𝜆ex Detection limit Reference
of sensors used for detection of
Anions ­(F− and ­OCl−) F −
46 20 𝜇 M ­CH3CN 490 nm –8
2.75 × ­10 M [138]
47 DMSO 542 nm 0.36 ppm [140]
48 DMSO, pH 8.0 510 nm 9.8 × ­10–9 M [141]
49 CH3OH/H2O, pH = 7.4 320 nm 0.025 × ­10–6 M [142]
OCl− 50 HEPES buffer, pH 7.0 325 nm 7.5 × ­10−7—1.1 × ­10−5 [147]
𝜇 mol/L
51 HEPES buffer 485 nm 6.68 × ­10–9 M [148]
52 CH3CN 523 nm 0.2 × ­10–6 M [149]
53 methanol–PBS, pH 7.4 510 nm 20 × ­10–6 M [150]
54 Tris–HCl/CH3CN buffer, pH = 7.4 480 nm 17.5 × ­10–9 M [152]
55a PBS 490 nm 0.21 μM [153]
55b 0.23 μM
56 DMSO, PBS, pH 7.4 530 nm 0.056 × ­10–9 M [154]
57 Tris – HCl, pH = 7.2 490 nm 0.023 × ­10–6 M [155]

13
 Journal of Fluorescence

Table 4  Comparison of results of different sensors discussed for the detection of small molecules
Analyte Probe Solvent 𝜆ex Detection limit Reaction time Reference

Thiols 58 HEPES buffer, pH 7.4 485 nm 9𝜇M 10 min [167]

59 H2O/DMSO 420 nm 0.026 𝜇 M 10 s [167]
60 PBS, pH = 7.4) containing 30% DMSO 454 nm 0.16 𝜇 M 10 min [168]
61a n -ethanol-phosphate buffer, pH 7.4 478 nm 77 nM 15 min [177]
61b 121 nM 10 min
62 HEPES buffer, pH = 7.4 550 nm - 25 min [178]
63 DMSO-PBS (pH 7.4) 538 nm 39.2 nM 20 min [178]
64 PBS-DMF, pH 7.4 608 nm 0.30 μM—Cys 10 min [181]
0.42 μM—Hcy
65 HEPES, pH = 7.4 492 nm 88 nM 20 min [182]
66 DMSO-PBS buffer, pH = 7.4 450 nm - 5 min [183]
67 CH3CN/H2O; PBS buffer, pH 7.4) 520 nm 0.12 μM 10 min [185]
68 EtOH-PBS, pH = 7.4) 525 nm 0.086 𝜇M 20 min [186]
CO 69 PBS buffer, pH 7.4, with 0.5% DMSO 490 nm 37 nM 15 min [191]
70 PBS buffer, pH 7.4, with 0.5% DMSO 490 nm 46 nM 20 min [192]
H2S 70 PBS/ ­CH3CN, pH = 7.2 495 nm - 10 min [204]
71 CH3CN:HEPES, pH 7.4 525 nm - 20 min [204]
72 PBS buffer:CH3COCH3, pH 7.4 450 nm 1 µM 15 min [205]

palladium in its various oxidation states. As the probe exhib- 7-hydroxy-4-methylcoumarin-8-carbaldehyde-(fluorescein)

its a fluorescence background or disturbance in signal, there
seems to be incorporation of electron withdrawing groups at
2’ and 7’ position in the probe 28. This showed an increase
in the ability of the xanthene core to act as a leaving group
in the ether cleavage releasing the fluorescent molecule giv-
ing a large increase in the fluorescence. This increase in the
rate of reaction proved to be a more useful palladium sensor
compared to other sensors. The size and shape-controlled
fluorescein modified nanoparticle probe 29 has proven to
be an effective sensor towards palladium detection. The low
detection limit as indicated in Table 1 makes it a better probe
for future research.
Detection of Zinc Ions
Zinc is the second most abundant transition metal in the
human body [95] and it helps in gene transcription, neural
signal transmission, mammalian reproduction, etc. [96, 97].
­ n2+ is still being explored. It can destroy neurons,
Toxicity of Z
glia and other cell types [98]. Usually most of the sensors
exhibited fluorescent enhancement through spirolactam ring
opening. But in probe 31, no ring opening was observed and
fluorescence enhancement was explained by CHEF effect.
­Zn2+ bonded with two oxygen atoms in the probe without
ring opening and a strong fluorescence was also observed.
The probe can be applied for the intracellular sensing [99].
Sensors based on fluorescein derivatives were gaining more
importance, but its coumarin conjugates were still unex-
plored. Jun-mei An et al. developed a Z ­ n2+ sensor (probe 32)
hydrazone which showed less interference from other transi-
­ n2+, ring opening occurred
tion metal ions. In this probe with Z
and ion bonded to the O of fluorescein moiety [100].
An azofluorescein sensor (probe 33) based on the PET
mechanism was explained on the basis of intramolecular
charge transfer of nitrogen lone pair electrons at the azo
bridge [101]. Another class of sensors was developed using
imidazole with fluorescein. Presence of pyridine-like nitro-
gen atom donors makes imidazole derivatives very good in
selective sensing of cations [102]. Balasubramanian Vidhya
et al. developed a sensor (probe 34) of fluorescein dye hold-
ing a imidazole moiety. Binding mechanism was explained
as ­Zn2+ is coordinated with the nitrogen from the imida-
zole, oxygen of amide and the imine nitrogen. This sensor
produced very good fluorescence and practical applicability
was proved with the sensing of ­Zn2+ ions produced during
apoptosis [103].
Fluorescent sensors based on Schiff bases are of high
demand because of the easy synthesis and also the nitro-
gen atom present increases the tendency towards binding
[104]. A Schiff base sensor (probe 35) with phenol func-
tionalized moiety and fluorescein hydrazine through Schiff
condensation method was reported in which fluorescence
enhancement was explained using chelation enhancement of
fluorescence and the PET mechanisms [105]. A fluorescein-
benzathiole sensor (probe 36) based on chelation enhance-
ment of fluorescence (CHEF) was developed for Z ­ n2+ ion
sensing. Coordination occurred with phenolic, and carbonyl
oxygen along with Schiff base group resulting in spirolactam

13
Journal of Fluorescence
ring opening. Sensor was found to be useful in experiment
with the test strips [106].
On comparing fluorescence spectra of the sensors, a 200
fold increase in fluorescence intensity is observed for sensor
34, which makes it a better turn-on fluorosensor. Quantum
yield value for the sensors 31, 32, 33, 35, and 36 are 0.03,
0.03, 0.64, 0.0258, and 0.241 respectively. With Zinc ion it
changes to 0.14, 0.28, 0.168, and 0.420 for sensor 31, 32,
35 and 36, respectively. Based on the given values probe 36
is found to produce the high intensity fluorescence. Also
for the probes 35 and 36, test strips were developed which
produced a bluish green and highly green fluorescence
respectively under UV light. Test strips are definitely an
add on advantage for the fluorescent sensors which makes it
more useful for direct qualitative estimation. Moreover, an
INHIBIT molecular logic gate was developed with two input
signals ­Zn2+ and EDTA, and output signal as the emission
intensity (Probe 35).
Detection of Cadmium Ions
Cadmium is an essential element and is commonly used in
agriculture, industries and many other fields [107, 108]. It is
highly toxic and can damage kidneys, bones, prostate cancer,
etc. [109–111]. Several sensors have been developed for the
detection of cadmium in various complex matrices.
Just like fluorescein the unique photophysical properties
of quantum dots (QD), increased its demand on construct-
ing sensors [112]. Thioglycolic acid (TGA) capped CdTe
QDs were modified using polyethylenimine which is fur-
ther combined with fluorescein isothiocyanate (FITC) by
Rijun Gui and coworkers (probe 37). The sensor worked
on the FRET mechanism for ­Cd2+ detection and was more
reliable since no intensity changes were observed from
excitation and emission light scattering [113]. DCF being
one of the effective derivatives of fluorescein, Xiao-yan Liu
et al. synthesized a derivative (probe 38) by choosing DCF
as the fluorophore and the diethylenetriamine as the iono-
phore with an acetic acid substituent. The ionophore bonded
effectively with ­Cd2+ to produce the fluorescence. Interfer-
ence produced by mercury ions was effectively masked for
the selective sensing of ­Cd2+ [114]. Diarylethene molecules
[115] are another class of great interest since it can combine
with other fluorophores and can exhibit very good fluores-
cent properties. Fluorescein-diarylethene derivative (probe
39) combined with another fluorophore quinolone can bind
with ­Cd2+ through oxygen atom of fluorescein, N atom of
quinolone and also ­ClO4− from the solvent (Fig. 7) [116].
Chemosensors developed for detection of C ­ d2+ is less
common due to the interference from other similar cations.
Probe 38 experienced significant interference from mercury
ions which was successfully eliminated on adding NaCl,
whilst probe 39 did not have much interference from other
cations. Probe 37 was able to reduce PL intensity fluctuation
and is more reliable. Ionophore diethylene triamine was used
in probe 38 which is successfully coordinated with cadmium
ions. Also pH does not affect diethylene triamine since the
pKa value for ionophore is low due to aromatization. While
in probe 39, a photochromic compound diarylethene was
bridged with fluorescein using quinolone. Diarylethene
molecules are gaining attention due to its photochemically
reversible and thermally irreversible character. Moreover,
the photochromic property in the sensor was used to develop
­ d2+ as input and fluorescence
a binary logic circuit with C
as output signals.
Detection of Magnesium Ions
Magnesium is the fourth most abundant cation in the human
body and has various functions in the human body includ-
ing skeletal development [117], DNA synthesis [118],
functioning of immune system and central nervous system.
Deficiency can cause health issues like coronary heart dis-
ease, diabetes, osteoporosis, etc. [119–121]. Different sen-
sors based on diverse fluorophores have been developed for
detecting magnesium ions.
Morpholine molecules have good ability to form com-
plexes with metal ions. DTAF-MOR (probe 40) was devel-
oped by linking morpholine with aminofluorescein using tria-
zine chloride (Fig. 8). Triazine chloride was chosen because
the three chlorine atoms present could easily undergo substi-
tution. Fluorescent enhancement of the probe occurred when
­Mg2+ bonded with the carbonyl group of the DTAF-MOR.
The probe was found very effective in sensing ­Mg2+ in the
biological system [122]. Chromone-based compounds are also
widely applied as fluorophores due to their excellent spec-
troscopic and pharmacological properties [123]. Chao-rui Li
et al. designed and synthesized a fluorescein derivative (probe
41) containing the chromone moiety as sensor for M ­ g2+. The
2+
sensor bound to M ­ g through a carbonyl group of both chr-
omone and fluorescein moiety along with one ethanol mol-
ecule and hydrogen oxide molecule [124].
A significant enhancement can be observed for the fluo-
rescence on adding the magnesium ion. Enhancement factor
for the probe 40 is 1.786. Fluorescence intensity for probe
39 increased on adding magnesium ion on the concentration
range 2 × ­10−7 –1.6 × ­10−6 mol/L and maximum enhance-
ment is observed at 1.2 × ­10−6 mol/L. For probe 41and 49,
58- fold increase was observed in the fluorescence. Concen-
tration range was between 0.05 – 0.40 µM. Quantum yield
and fluorescence lifetime for sensor 40 is 0.294 and 1.944 ns
respectively. Based on the pH effect on fluorescence change,
­pKa value of 40 was found to be 6.94, which is due to the
strong electron donating ability of the morphine group in
13

the sensor. Interference studies showed that the probe 41 can
show a relatively weak fluorescence for zinc ions as well.
Experiments were conducted to determine the limits of
detection and binding constant as given in Table 1 to prove
the effectiveness of the probe towards various metal ions.
From probe 1–16, probe 13 exhibited the lowest detection
limit and the value indicated that the probe is best suited to
detect ­Cu2+ ions at a minimum concentration. There was
no significant change in fluorescence observed in presence
of other metal ions but addition of copper changed fluo-
rescence intensity with the color changes demonstrating its
sensitivity. The highest binding with ­Cu2+ was exhibited
by probe 7. The absorption and fluorescence spectra of the
probes were investigated by mixing with various metal ions.
UV–Vis spectra of the probes containing copper ions range
from 150–500 nm.
Probes 17–22 showed a good selectivity to H ­ g2+ without
much interference from other ions. Studies showed that the
optimum pH range for smooth functioning is 4–10, but best
performance was observed in a slightly alkaline medium. A
bright fluorescence was obtained for every sensor on adding
­Hg2+. Detection limit shows that all these probes could sense
­Hg2+ in µM level and it makes the sensors more suitable for
identifying the ­Hg2+ content in the environment. Among the
discussed, probe 19 has the lowest detection limit.
For ­Fe3+ the probe containing benzothiazole (probe 23)
exhibited the detection limit at 1.085 × ­105 which is lower
compared to all other probes. The binding property of cat-
echol dye with F­ e3+ showed higher binding constant. Differ-
ent color changes were observed in addition of F ­ e3+ ions to
sensor and there was no significant change in fluorescence in
presence of other metal ions. UV Vis spectra of the probes
range from 300-400 nm.
The probe 27 exhibited the lowest detection limit com-
pared to other probes for palladium and the value indicated
that the probe is best suited to detect the palladium ions at
a minimum concentration. There was no significant change
in fluorescence observed in presence of other metal ions but
addition of palladium showed a bright green fluorescence.
UV Vis spectra of the probes ranges from 400-500 nm.
Probes 31–36 were effective in sensing the ­Zn2+ with-
out much interference. Experimental results show that a
slightly alkaline medium is more favoured. A bright green
fluorescence was observed on adding ­Zn2+ to the sensor.
Using Job’s plot, the binding constant of each probe is cal-
culated. Probe 33 shows good binding properties with Z ­ n2+.
Detection limit value shows that probe 31 is very effective in
detecting ­Zn2+ even at low concentration level.
Probes 37–39 shows a strong fluorescence with C ­ d2+
with not much interference. Excitation wavelength is at
470 nm, 470 nm and 350 nm for probes 37, 38 and 39
13
Journal of Fluorescence
respectively. Detection limit value shows that µM level
detection is possible with C ­ d 2+. Practical applications
involve bioimaging and moreover the sensor can be used
for developing a logic circuit (probe 38).
Both the probes 40 and 41 were successful in real time
monitoring of ­Mg2+ level. A strong fluorescence with an
obvious colour change is observed on adding M ­ g2+ for the
sensors on fluorescence and UV- visible studies. Compara-
tive interference from other ions were absent for these sen-
sors which were able to detect ­Mg2+ in µM level.
Fluorescein Based Fluorescent Probes
for Simultaneous Detection of Two Cations
Currently, the design of fluorescent probes with multi ion
responses has gained increasing attention. In fluorescein based
schiff base (probe 42) monoaldehyde-(2-pyridyl) hydrazone
­(H2L), the fluorescence emission was observed due to bind-
ing of ­Cu2+ and A ­ l3+ through phenolic oxygen, imine and
pyridine N atoms with suppression in C = N atoms leading to
change in fluorescence outlining its sensitivity [125].
Report exists in the literature for a multiple fluorescein
based (probe 43) FHCS molecule by combining fluorescein,
hydrazone, cyanuric chloride and salicylaldehyde chromone
for simultaneous determination of ­Al3+ and ­Zn2+. UV–Vis
spectra of FHCS molecule observed due to the presence of
carbonyl C = O, aromatic ring and C = N absorption band of
fluorescein and Schiff base whose absorption band changed
after addition of ­Al3+ and ­Zn2+ [126].
Prabhjot Kaur et al. synthesized an “on–off” system
for the detection of Cd(II) and Co(II) metal ion using
1,2,3-triazole linked fluorescein chemosensor (FLT)
(probe 44). The 1,2,3-triazole-linked fluorescein (FLT)
was synthesized by 1,3-dipolar cycloaddition of which
contains biomolecular alkyne units with benzyl azide. The
presence of N donor sites creates an excellent framework
for ­Cd2+ and C ­ o2+ which can be used for their detection
[127].
A novel phenolphthalein-fluorescein dye derivative (PF)
(probe 45) was synthesized using a solution of dialdehyde
derivative of phenolphthalein 1 and fluorescein hydrazide
2 (Fig. 9). The binding of ­Hg2+ and ­Zn2+ led to the formation
of ring opened amide form of PF [128].
Probe 42 in the presence of ­Cu2+ and ­Al3+ showed a fluo-
rescence emission peak at 520 nm with a fluorescent-yellow
color, while probe 43 showed a fluorescence peak at 384 nm
with fluorescent blue color. Probe 44 observed to show emission
peak at 456 nm and probe 45 at 521 nm with fluorescent- yellow
color. The experiments were conducted using ethanol as sol-
vent. Table 2 shows comparison of results of different samples
used in the detection of two cations.
Journal of Fluorescence
Fig. 1  Structure of Xanthene dye
Fluorescein Based Fluorescent Probes
for Detection of Anions
Anions have a vital role in the functioning of biological
systems. Compared to cations, selective sensing of anions
is difficult. Large size, low charge to size ratio and different
shapes of anions are some of the challenges faced while
designing chemosensors. Two main strategies are involved
for anion sensing:
i. Using positively charged receptors
ii. Using neutral ligands
Binding between an anion and sensor occurs through
electrostatic interactions or hydrogen bonding [129]. When
binding occurs specific signals are produced by the chem-
osensor. Schematic representation of how anion sensing
works is shown in the (Fig. 10) [130].
It was found that –N containing moieties like urea, thio-
urea, amides, etc., when introduced into fluorescein can
induce effective sensing of anions using the spirolactam
ring opening [130].
Detection of Fluoride Ion
Concentration of the fluoride ion decides whether it is dan-
gerous or useful to the humans [131]. Appropriate levels of
fluoride ion are beneficial to dental care and osteoporosis.
Fig. 3  Fluorescein open-closed
structure
Fig. 2  Modification sites in Fluorescein molecule
According to WHO 1.5 mg L ­ −1 is the maximum F ­ − limit that
can be present in drinking water [132]. Community water
fluoridation is an effective technique used in different coun-
tries to make sure that fluoride deficiency is not observed in
people [133]. Several fluorescent probes have been devel-
oped based on different fluorophores (Fig. 11) [134, 135].
A fluorescein sensor using NBS as a masking agent
(probe 46) was prepared by modifying a sensor previously
prepared by Yang et al. [137]. The sensing of ­F− is explained
as a two-step process- adding of F ­ − deprotects NBS and
produces a monoionic form of fluorescein which further
undergoes deprotonation on addition of F ­ − which produces
the diionic form of fluorescein which is highly fluorescent
[138]. Competition from water has been observed in few of
the developed sensors [139]. Ping Zhang et al. addressed
this issue by combining fluorescein with antipyrine so that
acidity of –NH group increases and can compete with water
molecules. The probe 47 bonded to F ­ − ion through –NH
and –COOH through hydrogen bonding [140]. Hong Yeong
Kim et al. developed a probe 48 from DCF by using dimeth-
ylphosphinyl group as –OH protecting group which could be
easily removed by fluoride ions (Fig. 12). Sensing of the probe
13

Fig. 4  Fluorescein probe 6 used
for ­Cu2+ detection." Reproduced
with permission from Ref. [55].
Copyright (2016) The Royal
Society of Chemistry."
occurs by cleavage of the protecting group on adding ­F− ions
and regenerates the DCF which accounts for the fluorescence
enhancement [141]. Probe 49 was developed by using fluo-
rescein and coumarin fluorophores. Tert-butyldiphenylsilyl
was selected as the functional group because of its reactivity
towards ­F− and also due to the high selectivity in the aque-
ous medium. Sensing occurred by breaking the O-Si bond
on adding the fluoride anion onto the sensor [142].
Most significant fluorescent enhancement is observed
for probes 46 and 48 with approximately 60 and 490-fold
increase in fluorescence, respectively. Not much interference
from other ions is faced by probes 46 and 49. Some interfer-
ence from acetate ions, H­ g2+ and C
­ u2+ ions respectively are
−
found for the detection of ­F ion which is masked effectively
for the detection (Probes 47 and 48). Test strips application
was also done to confirm the practical effectiveness (Probe
46).
Detection of Hypochlorite Anion
Hypochlorite is used as a disinfectant in drinking water,
bleaching agent, etc. Excessive intake can cause tissue dam-
age, liver injury, arthritis and cardiovascular diseases [143,
144]. Different fluorescent sensors have been developed for
the hypochlorite sensing [145, 146].
Fig. 5  Fluorescein probe 19
used for ­Hg2+ detection, "
Reproduced with permission
from Ref [75], Copyright (2017)
Elsevier."
13
Journal of Fluorescence
Fang-Jun Huo et al. developed a probe 50. In presence
of ­ClO−, ­Cu+ oxidizes to ­Cu2+ which forms a complex with
FHP and ring opening occurs inducing a strong green fluores-
cence (Fig. 13). It is the first fluorescein sensor to be reported
of HOCl/ClO− in water [147]. In the same year Yi Zhou
et al. also developed three probes 51 based on dihydrofluo-
rescein in 100% aqueous media. The probe detected HOCl by
specific oxidation reaction which switched probe from non-
fluorescent form to fluorescent conjugated form. Among the
three sensors FCN2 had lower detection limit, fast response
time and moreover it could estimate the HOCl accumulation
in living cells [148]. HOCl being closely related to ­H2O2 it
was really important to selectively detect HOCl in presence
of ­H2O2. A dual lock sensor FBS was developed by combin-
ing xanthenone derivative with boronic ester and thiolactone
(probe 52). FBS reacting with H ­ 2O2 and other ROS yields
FS which is also non-fluorescent. Only with O ­ Cl−, FBS
converted to fluorescent fluorescein. This dual lock method
reduced the interference from other ROS [149].
With probe 53, ­OCl− forms delocalized xanthane of fluo-
rescein moiety which accounts for the fluorescent enhance-
ment. S and O heterocycles in fluorescein are the reason
for its high selectivity of ­OCl−. The sensor can be success-
fully used for pathogen analysis and ­OCl− based disinfec-
tion in drinking water. Compounds with unbridged C = N
Journal of Fluorescence

­ e3+detection," Reproduced with permission from Ref [84], Copyright (2019) The Royal Society of Chem-
Fig. 6  Fluorescein probe 25 used for F
istry."

(non-fluorescent) shows fluorescence when C = N isomeri-
zation is inhibited. HOCl can oxidise the C = N to carbonyl
groups [151]. Based on this property Jie Li et al. developed
a fluorescent sensor (probe 54) in which when O ­ Cl− is
added, F-BH converts to formylated fluorescein and C = N
isomerization is inhibited which results in strong fluores-
cence [152]. Formylated fluorescein was found to possess
very good fluorescent properties and therefore many sensors
were developed based on them. Hypochlorite sensors have
been developed (probe 55a- FN-1 and probe 55b- FN-2)
by condensation between the aldehyde group of fluorescein
and amine group of diaminonaphthalene. ­OCl− can break
the C-N bond and collapse the PET mechanism between
fluorescein and diaminonaphthalene moieties which lead to
the fluorescence enhancement [148].
A probe has been developed (FCZ) by combining fluo-
­ Cl− which
rescein and carbazole (probe 56) for detecting O
could break the C = N bond in FCZ and open the ring pro-

Fig. 7  Fluorescein probe 39 used for ­Cd2+ detection,," Reproduced with permission from Ref [116], Copyright (2016) Elsevier."

13

ducing carboxyl anion. Chromatographic studies confirmed
­ Cl− producing the fluorescein [153].
that FCZ reacted with O
In probe 57, the reaction mechanism proceeds through an
oxidation reaction favoured by the hypochlorite. –NH 2
group in the probe was oxidised to the imino group and
the –CH = ­NH2 attributes to the fluorescence enhancement
[154].
Comparing the spectral values of the sensors, it indicates
that sensors 51, 53, 54, and 57 shows 1643.4, 586.8, 18,
and 150 fold increase in the fluorescence enhancement,
respectively. Quantum yield of the sensors 51, 53, & 55
Fig. 8  Fluorescein probe 40 used for ­Mg2+ detection, " Reproduced with permission from Ref [122], Copyright (2015) Wiley."
13
Journal of Fluorescence
are 0.71, 0.82, and 0.363, respectively. Based on quantum
yield value, sensor 53 will be producing stronger intensity
on adding hypochlorite ions. The fluorescence intensity and
­OCl− concentration for all sensors show a very good lin-
ear relationship with a correlation coefficient value 0.99.
Using zebrafish and HeLa cells, biological applications were
carried out. Drosophila gut system is known for producing
HOCl. Sensor 52 was able to produce a green fluorescence
to the gut ingested with bacterial extracts which implies that
it can be used for signalling bacterial attacks. Disinfection
effect of ­OCl− in bacteria and bacterial films was studied
Journal of Fluorescence
Fig. 9  Fluorescein probe 45 used for ­Hg2+ and ­Zn2+ detection, " Reproduced with permission from Ref [128], Copyright (2017) Elsevier”
by using Rhodobacter sp. strain SW2 (Probe 53). Practical
applicability of probes on real samples was also determined
by using them on tap water and NaClO disinfectant (probes
50 and 57).
All the ­F− probes had a very low detection limit rang-
ing from micromolar to nanomolar which makes the sen-
sors more applicable in testing F­ − in water bodies especially
for drinking water. pH studies show that the sensors favour
slightly alkaline condition. Results from competition experi-
ments, cytotoxicity studies, etc., favour the application of
these sensors.
Hypochlorite detection was observed with a strong fluo-
rescence with much less interference. pH ranging from 4–9
can be used, but the most suitable value was observed at pH
7.4. Detection limit of all the discussed probes varied from
micromolar to nanomolar which makes these sensors practi-
cally applicable. Cytotoxicity results also confirmed that the
sensors are safe to use. Table 3 shows comparison of results
of sensors used for detection of anions ­(F− and ­OCl−)
Fig. 10  Mechanism of anion
sensing. “ Reproduced with
permission from Ref [130],
Copyright (2017) Elsevier.”
Fluorescein Based Fluorescent Probes
for Detection of Small Molecules
In this section we are discussing the sensing of gasotrans-
mitters and various thiols. Compared to ions the sensing
mechanism for biomolecules is quite complicated. Due to
the intrinsic properties and the highly complex structure, the
strategies used for the sensing differs greatly. Use of fluo-
rescence spectroscopy has an advantage over other conven-
tional methods since it can be used for real time sensing of
the tissues without damaging them. It helps in understanding
different physiological and toxicological properties of the
biomolecules.
Detection of Thiols
Thiols are a class of organic compounds containing sulf-
hydryl groups, i.e., S and H atoms attached to the carbon
atom [154]. Biological thiols like cysteine (Cys), homocyst-
13

Fig. 11  Graphical representation of fluoride sensing using fluores-
cent probe. “ Reproduced with permission from Ref [136], Copyright
(2016) The Royal Society of Chemistry."
eine (Hcy) and glutathione (GSH) have many physiological
importance [155]. Thiol containing molecules can be used
as drugs against pulmonary diseases [156], oxidative stress,
[157, 158] and inflammations [159]. All these molecules
have specific roles in the smooth functioning of the body.
One important function of GSH is to store cysteine which
is important for glutathione synthesis. GSH can be used as
biological markers [160]. Deficiency of Cys causes various
symptoms like hair pigmentation, skin lesions, growth retar-
dation, liver damage, etc. [161]. Excess of Cys can cause
AIDS, Alzheimers and cardiovascular diseases [162, 163].
Detection of thiols requires efficient techniques because all
the biothiols are homologous and have similar properties.
Several fluorescent sensors using different fluorophores were
synthesized [164, 165].
An iminofluorescein-Cu2+ sensor (probe 58) has been
reported for thiol detection. On adding the thiol, C­ u2+ got
2+
removed from the iminofluorescein-Cu complex and fur-
ther hydrolysis yielded fluorescein- aldehyde which accounts
for fluorescence enhancement [166]. Many sensors have
been developed using fluorescein as the fluorophore and
introducing maleimide units through 1,2-Michael addi-
tion for thiol detection. Based on this Fangjun Huo et al.
developed a sensor (probe 59) using fluorescein and maleic
anhydride. Maleimide ring attached weakens the fluores-
cence of the sensor. When thiol is added, it saturated the
Fig. 12  Fluorescein probe 48
used for ­F− detection, “ Repro-
duced with permission from
Ref [141], Copyright (2015)
Elsevier”
13
Journal of Fluorescence
C = C bond and fluorescence got enhanced [167]. Yunchang
Liu et al. developed a fluorescein sensor with a thiol selec-
tive 2,4-dinitrobenzenesulfonyl (DNBS) group (probe 60)
(Fig. 14). When connected with DNBS, no fluorescence or
increase in absorption band is observed due to the spirocy-
clic form. When thiol was added DNBS group was removed
resulting in fluorescein with high fluorescence [168]. Further
sensors were developed for detecting Cys, Hcy and GSH.
Cysteine specific probes are developed on five different strat-
egies [169]:- cyclization with aldehydes [170], Michael addi-
tion [171], addition-cyclization with acrylate [172], chemical
ligation, [173] and aromatic substitution followed by rear-
rangement [174].
Sensors based on acrylate moiety have been developed
which can be differentiated between Cys and Hcy [175].
Hulin Wang et al. developed two fluorescein probes 61a and
61b with a single and double acrylate respectively. When
thiol was added to the acryloyl moiety, ester cleavage took
place producing fluorescein molecules, which accounts for
the fluorescence enhancement. Comparing the two probes,
double acrylate containing sensor had more selectivity
[176].
By condensing acryloyl chloride with seminapthofluo-
rescein probe 62 (Seminaphthofluorescein bis-acrylate) has
been developed. On adding Cys conjugate addition occurs
followed by rapid cylcization to release SNF which has a
strong fluorescence. With GSH and Hcy, addition occurs
but cyclization does not happen and probe still exist in non-
fluorescent form [177]. Yuan Ji et al. developed another
sensor (probe 63) using acrylate moiety with fluorescein
and julodine. A PET mechanism was observed between pi
electrons transfer of LUMO of acryloyl group and HOMO of
julodine moiety leading to fluorescence quenching. Fluores-
cence enhancement was observed by inhibition of the PET
process [178].
Near Infra-red sensors [179, 180] have more possibilities
in bioimaging sensors compared to non-NIR ones. Based
on this a sensor (probe 64) has been developed based on
naphthofluorescein modified by chloroacetate. Chloroacetate
reacts with the sulphur containing moiety of Hcy and Cys
to form thioethers followed by cyclization to produce the
NIR and highly fluorescent naphthofluorescein (NF) [181].
Hailang Chen et al. developed a sensor (probe 65) using
fluorescein and rhodamine-B with maleic anhydride. The
probe TRDM-F on reacting with Cys undergoes Michael
addition followed by cyclization releasing the fluorescein
Journal of Fluorescence
Fig. 13  Fluorescein probe 50 used for ­OCl− detection, “ Reproduced with permission from Ref [147], Copyright (2012) Elsevier”
molecule by O–O breakage with strong fluorescence [182].
Zhen-A fluorescent sensor (probe 66) was reported based
on a hybrid of fluorescein and coumarin fluorophores. First,
a fluorescein-coumarin hybrid was developed which was
further treated with acryloyl chloride to modify the other
hydroxyl group. When Cys was added, conjugate addition
occurs with spirolactam ring formation. Quinone structure of
the sensor attributes to the fluorescence enhancement [183].
Disulfide bonds are used as linkers in fluorescent sensors
due to their affinity towards reductive thiols [184]. A probe
with disulfide linkage with fluorescein and acenaphthene
(probe 67) was reported for the detection of GSH or Cys
and when these groups were added disulfide bond cleaves
and attack occurs at carbonyl groups leading to formation of
fluorescein moiety with strong fluorescence [185]. Further
the probe 68 was developed by combining fluorescein and
acenaphthene. Acrylate part of probe undergoes addition
with thiol followed by an intramolecular cyclization reac-
tion with ester cleavage to produce the strong fluorescent
fluorescein moiety (Fig. 15) [186].
Fluorescence spectra of probes 63, 65, 66, 67 and 68 on
adding the particular thiol shows a 30, 95, 62.4, 10, and
18-fold increase in the fluorescence enhancement respec-
tively. Quantum yield value for probes 64, 66, 67 and 68 was
found to be 0.008, 0.007, 0.004 and 0.003, respectively. On
Fig. 14  Fluorescein probe
60 used for thiol detection, “
Reproduced with permission
from Ref. [170], Copyright
(2016) Elsevier”
adding the thiol, quantum yield of these sensors increased to
0.020, 0.60, and 0.61 for 66, 67 and 68. The values obtained
for probes 67 and 68 are close to the quantum yield value of
fluorescein (Φ = 0.63). Fluorescence titration value showed
that the fluorescence intensity of probes 61, 63, 64, 65, 66,
and 68 have a linear relationship to the concentration in
the range of 0–500 nM, 0.3–0.30 M𝜇, 1–40 M𝜇, 0–50 M𝜇,
0–10 M𝜇, and 0–10 M𝜇 respectively. Other than the normal
detection applications sensor 62 was successful to determine
the Cys content in human plasma which suggests it as a
potential detector for quantitative detection of Cys content
in the biological systems.
Detection of Carbon Monoxide
CO was always considered as an unimportant molecule till it
was very lately identified as a mediator in cellular functions
and biological importance [187]. CO played an important
role in the inflammatory and cardiovascular diseases and
therefore it was used as an important molecule in the field
of drug designing [188]. Pd -mediated Tsuji–Trost reaction
mechanism is usually observed for developing CO sensors
[189]. Since CO molecules cannot be used directly for the
studies, an alternative CO releasing molecule CO-RM-3 is
used [190].
13

Fig. 15  Fluorescein probe 68 used for thiol detection, “ Reproduced with permission from Ref. [187] Copyright (2016) Elsevier”
Weiyong Feng et al. developed an allyl chloroformate
mediated fluorescein FL-CO-1 (probe 69) with P ­ dCl2 as an
additive trap for CO. FL-CO-1 is non-fluorescent with P ­ d2+
0 2+
while shows fluorescent properties with ­Pd . ­Pd is reduced
by CO to produce P ­ d0 which promotes Tsuji-Trost reaction
which releases the highly fluorescent fluorescein molecule
[191]. Due to the instability of the allylcarbonate protec-
tion group, the probe FL-CO-1 was found to be less stable.
So they further modified the probe with allyl ether as the
protection group to develop probe 70a and 70b. Here also
the allyl group can be removed by ­Pd0-mediated Tsuji-Trost
reaction (Fig. 16) [192].
Fluorescence spectral studies of the sensors show that
sensor 69 and 70 can produce a 100 and 150 fold increase
in fluorescence enhancement, respectively. Both the sensors
produce a strong yellow green fluorescence which is very
evident even in mild conditions which makes both the sen-
sors naked eye detectors for CO. Concentration dependent
Fig. 16  Fluorescein probe 70
used for CO detection, “ Repro-
duced with permission from
Ref. [193] Copyright (2017)
American Chemical Society”
13
Journal of Fluorescence
fluorescence spectra for 69 have a correlation coefficient
value of 0.99856. Various reports showed that heme can
produce heme oxygenase (HO)-derived CO in living cells.
Both the sensors were able to detect the endogenous CO
produced in the cells preincubated by heme.
Detection of Hydrogen Sulphide
H2S was also initially considered only as a toxic gas, and
it was very recently identified as the third most important
gasotransmitter and also its various physiological roles like
gastrointestinal functions [193]. It is also used as a signalling
molecule in cells, nervous system, circulatory system, etc.
[194]. Even though it plays an important role, it is equally
dangerous to the human body [195, 196]. It can damage nor-
mal functioning of the body and cause diseases like Asthma
[197]. Cardiovascular diseases [198], inflammations [199].
It is important to monitor the hydrogen sulphide in the
Journal of Fluorescence
Fig. 17  Fluorescein probe 71
used for ­H2S detection, “ Repro-
duced with permission from
Ref. [204] Copyright (2017)
American Chemical Society”
biological system. Fluorescent sensors for H­ 2S are developed
mainly on three approaches azide reduction [200], nucleo-
philic addition [201], and copper sulfide precipitation [202].
Several sensors were developed based on this mechanism.
A chemosensor has been reported (probe 71) based on
the fluorescein and hydroxyquinoline which can bind with
­Cu2+. When C ­ u2+ is added fluorescence quenching happens
to the sensor on forming a complex with the copper ion.
When sulphide ion is added it combines with the copper and
fluorescence revival occurs (Fig. 17) [203].
A fluorescein sensor has been developed by protecting
both hydroxyl groups with dinitrophenyl ether (probe 72).
When ­H2S reacts with probe it removes the dinitrophe-
nyl ether in subsequent steps to produce fluorescein in its
quinoid form, which ascribes to the fluorescence and later
converts to the lactone form due to the aprotic solvent ace-
tonitrile used [204]. The sensor developed by protecting
hydroxyl group using thiophene carbonyl group (probe 73)
acts as a probe for ­H2S. When ­H2S is added C-O bond is
cleaved and thiophenylcarboxylate leaves forming fluores-
cent fluorescein molecules [205].
Probe 71 was designed using a displacement reaction
where copper ions are displaced by sulphide ions. Quan-
tum yield of the sensor was 0.123 which on interaction with
copper reduced to 0.061 due to the copper complex formed
and on adding the sulphide ions an enhancement occurs and
final quantum yield value will be 0.132. For probe 72 the
quantum yield value was found to be 0.564. Fluorescence
titration showed the linear relationship between fluorescence
intensity and the concentration in the range of 0-140 M and
20-100 M for 72 and 73. Probe 73 was able to do more appli-
cation studies than the other two sensors. Fast response time
of the sensor was confirmed by using the sensor on com-
mercially available ovine plasma. Using T cells & natural
killer cells exogenous, and using living HepG2 and living
Raw 264.7 macrophage cells endogenous ­H2S detection was
studied. Finally bioluminescence imaging of exogenous and
endogenous ­H2S was done on living mice. Studies indicate
that the sensor 73 is highly capable in detecting the H ­ 2S in
the biological system and by understanding the morphological
changes various diseases can be diagnosed in the earlier
stage. Table 4 shows comparison of results of different sen-
sors discussed for the detection of small molecules.
Probes 58–60 were used for thiol detection, and remain-
ing probes 61–68 were used for selective detection of Cys,
Hcy and GSH. Strong fluorescence was observed without
any interference from other reactive species or amino acids.
The probes were able to detect between nanomolar to micro-
molar range which makes the probe effective in applying in
biological systems. Probe 59 shows a very fast response time
(10 s) compared with other probes which needs 5–25 min to
complete the reaction. Both the probes have a strong fluo-
rescence with CO and excitation wavelength at 490 nm. It
can detect CO in nanomolar concentration. Probes work best
in a slightly alkaline medium (pH = 7.4). Probe 69 and 70
take 15 and 20 min, respectively to complete the reaction.
Competition, cytotoxicity and binding properties confirmed
these can be used effectively for CO detection. Probes 70–72
are used effectively for ­H2S detection. They show a strong
fluorescence with ­H2S with less interference. Probes can
sense ­H2S in micromolar level and work effectively in a
slightly alkaline medium. Response time for the probes is
between 10–20 min.
Conclusion
The past decade has seen a huge revolution in the field of
sensing using fluorescent sensors. This article discussed the
structural modifications of fluorescein molecules for analyte
sensing over the last ten years. It was observed that the spiro-
lactam structure of fluorescein favoured a lot in developing
sensors. Fluorescein molecule were combined with other
fluorophores and ratiometric sensors were also developed.
All these fluorescein derivatives display wide application in
biological experiments where they can covalently attach to
several functional moieties such as peptides, proteins, nucle-
otides, drugs, hormones and lipids via phenyl ring. It also
helped in sensing the toxic species in biological systems.
All the discussed sensors are highly selective and moreover
13

possessed favourable features which made it easier to apply
in the biological system. Also the majority of the sensors
worked as naked eye sensors which made them user friendly.
The approach of using fluorescein derivatives can be
employed as a strategy for the design of novel biosensors
and glycosides in the coming future. The structural modifi-
cation in the fluorescein moiety led to formation of differ-
ent fluorescein-based probes as common reagents which has
proven to be a sensitive and specific analytical tool in life
sciences and also in the replacement of radioisotope applica-
tions. In addition to their high yield synthesis and efficient
separation, these fluorescein probes will certainly attract
more attention in the future and can replace other probes for
detection of analytes.
Fluorescein molecules have some disadvantages like all
solvent systems cannot be used with them and also using
organic solvents can be dangerous for some biological sys-
tems. It is also observed that the number of sensors developed
for metal ions were comparatively higher compared to other
molecules. Therefore, future studies could be more oriented
in developing sensors which can be used for other small mol-
ecules. Similarly, single species sensors are more common
compared to multianalyte sensors. Sensing more species by
making small changes in reaction conditions or introducing
groups which can selectively sense analytes, with the same
sensor, will be some of the areas to be explored. Since the
mechanism of sensing is very similar it is very easy to modify
the already known sensors with different functional groups
or fluorophores with improved properties.
Authors’ contributions Keerthana S Lieterature data collection; Bincy
Sam: Interpretation and writing; Suhakar YN: Critical analysis; Louis
George: Reviewing and Editing; Anitha Varghese: Reviewing and
Editing.
Funding This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Declarations
Conflict of Interest The authors declare that they have no competing
interests.
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