Volume 46 Number 23 7 December 2017 Pages 7097–7472

Chem Soc Rev

Chemical Society Reviews
rsc.li/chem-soc-rev

ISSN 0306-0012

TUTORIAL REVIEW
Thorfinnur Gunnlaugsson, Engin U. Akkaya, Juyoung Yoon,
Tony D. James et al.
Fluorescent chemosensors: the past, present and future
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Fluorescent chemosensors: the past, present

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and future
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46, 7105
Di Wu,a Adam C. Sedgwick, b Thorfinnur Gunnlaugsson, *c Engin U. Akkaya, *d
Juyoung Yoon *a and Tony D. James *b

Fluorescent chemosensors for ions and neutral analytes have been widely applied in many diverse fields
such as biology, physiology, pharmacology, and environmental sciences. The field of fluorescent
chemosensors has been in existence for about 150 years. In this time, a large range of fluorescent
chemosensors have been established for the detection of biologically and/or environmentally important
species. Despite the progress made in this field, several problems and challenges still exist. This tutorial
review introduces the history and provides a general overview of the development in the research of
fluorescent sensors, often referred to as chemosensors. This will be achieved by highlighting some
Received 12th August 2017 pioneering and representative works from about 40 groups in the world that have made substantial
DOI: 10.1039/c7cs00240h contributions to this field. The basic principles involved in the design of chemosensors for specific
analytes, problems and challenges in the field as well as possible future research directions are covered.
rsc.li/chem-soc-rev The application of chemosensors in various established and emerging biotechnologies, is very bright.

Key learning points

(1) Strategies for the design of fluorescent chemosensors
(2) Sensing mechanisms involved in the design of fluorescent chemosensors
(3) Applications of fluorescent chemosensors
(4) Usefulness of fluorescent chemosensors for in vitro and in vivo studies
(5) Key problems and challenges in the field of fluorescent chemosensors

1. Introduction F. Goppelsröder in 1867, and was a method for the determina-

tion of aluminum ion (Al3+) by forming a strongly fluorescent
Compounds incorporating a binding site, a fluorophore, and a morin chelate.2 This led to the development of a number of
mechanism for communication between the two sites are called fluorescent chemosensors for the determination of many other
fluorescent chemosensors.1 If the binding sites are irreversible metal ions, over the subsequent several decades, marking in
chemical reactions, the indicators are described as fluorescent part the birth of analytical chemistry as we know it. In fact, the
chemodosimeters. These two definitions as well as the term early fluorescent chemosensors, concentrated mainly on the
‘‘fluorescent probe’’ have been used interchangeably and ambi- detection of metal ions rather than the detection of anions or
guously over the past few decades therefore we have unified the neutral species. This is due to the selective binding of metal
area to describe them all as fluorescent chemosensors in this ions in water being significantly easier than that of anions or
review. The first fluorescent chemosensor was reported by neutral species. However, more recently and since around 1980,
we have witnessed an explosive growth and development of the
a
Department of Chemistry and Nano Science, Ewha Womans University,
area catalyzed by the inspirational and pioneering work by the
Seoul 120-750, Korea. E-mail: jyoon@ewha.ac.kr two fathers of modern chemosensors: de Silva and Czarnik.3
b
Department of Chemistry, University of Bath, Bath, BA2 7AY, UK. Since those pioneering days, fluorescent chemosensors have been
E-mail: t.d.james@bath.ac.uk extensively developed and the scope of applicability extended to
c
School of Chemistry and Trinity Biomedical Sciences Institute (TBSI),
include numerous biologically important analytes. In particular,
Trinity College Dublin, The University of Dublin, Dublin 2, Ireland.
E-mail: gunnlaut@tcd.ie
fluorescent chemosensors for biologically and/or environmentally
d
UNAM-Institute of Material Science and Nanotechnology, Bilkent University, important cations, anions, small neutral molecules as well as
Ankara 06800, Turkey. E-mail: eua@fen.bilkent.edu.tr biomacromolecules (such as proteins and DNA) have been

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developed along with a rapid advancement in microscopic fluorescence (CHEF),2 intramolecular charge transfer (ICT),3
imaging technologies. Analyte detection by a fluorescent chemo- photoinduced electron transfer (PET),4 aggregation induced emis-
sensor is usually achieved through one or more common photo- sion (AIE)5 and the number of approaches is still expanding.
physical mechanisms, including chelation induced enhanced Due to the high levels of sensitivity and in particular their

Di Wu was born in Hubei Adam C. Sedgwick graduated with

province, P. R. China, in 1987. a 1st class MChem in Chemistry
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

He received his Bachelor degree for Drug Discovery from the Univer-
(2010) and PhD degree (2015) sity of Bath in 2014. During his
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from Central China Normal Univer- undergraduate degree he under-

sity (CCNU) under the supervision took an industrial placement at
of Professor Sheng Hua Liu and BioFocus (Now Charles River)
Jun Yin. Subsequently, he joined working as a medicinal chemist
Prof. Juyoung Yoon’s research synthesizing compound libraries for
group at Ewha Womans University various drug discovery applications.
(South Korea) as a postdoctoral He is currently working towards his
fellow. His research interests focus PhD at the University of Bath
Di Wu on fluorescent chemosensors and Adam C. Sedgwick developing novel sensors for the
new organic functional materials. detection of reactive oxygen species.

Thorfinnur (Thorri) Gunnlaugsson Engin U. Akkaya is a Professor at

MRIA, is a Professor of Chemistry Bilkent University, Department of
in the School of Chemistry, Trinity Chemistry and UNAM-National
College Dublin (TCD). His research Nanotechnology Research Center.
interests lie broadly within the He is a Fellow of the Royal Society
fields of medicinal, organic, of Chemistry, and a member of the
inorganic supramolecular and Science Academy (BA) of Turkey.
materials chemistries. He is a His research interests include
Fellow of TCD, and was elected photodynamics, molecular logic
as a Member of The Royal Irish gates and molecular devices, and
Academy in 2011. In 2014, he as information processing therapeutic
awarded The Institute of Chemistry agents. He authored 105 papers
Thorfinnur Gunnlaugsson of Ireland (ICI) Annual Award for Engin U. Akkaya and has an H-index of 46.
Chemistry (Eva Philbin Lecturer).
He is the author of over 220
papers and has an H-index of 70.

Juyoung Yoon is currently Tony D. James is a Professor at

Professor of Department of the University of Bath and Fellow
Chemistry and Nano Science in of the Royal Society of Chemistry
Ewha Womans University. His and holds a prestigious Royal
research interests include investi- Society Wolfson Research Merit
gations of fluorescent chemo- Award In 2013 he received the
sensors, molecular recognition, Daiwa-Adrian Prize and in 2015
and new organic functional the Inaugural CASE Prize. His
materials. He has published over research interests include many
280 SCI research papers with aspects of Supramolecular chem-
h-index of 82. istry, including: molecular recogni-
tion, molecular self-assembly and
Juyoung Yoon Tony D. James sensor design. He is the author
of over 232 papers and has an
h-index of 58.

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ability to be used for temporal and spatial sampling for in vivo

imaging applications, fluorescent chemosensors have been
widely applied in a variety of fields such as biology, physiology,
pharmacology, and environmental sciences. With the advent of
two or multi-photon excitation and high and super-resolution
fluorescence microscopy we will see an ever increasing need for
highly sensitive and selective chemosensors for in vivo biological
applications.
There are a number of reviews that have been compiled
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describing fluorescent chemosensors, however, to the best of

our knowledge, most of these reviews focus on either diverse
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fluorochromes or analytes. Only a few reviews have taken a step

back and carried out an overview of progress of this field.6,7
This review summarizes progress in the development of fluo- Fig. 1 Structures of the fluorescent chemosensors 1–3.
rescent chemosensors over the last 50 years and introduces
representative chemosensors for the detection of cations,
anions, small neutral molecules as well as biomacromolecules. quantum yield, and a substantial decrease in phosphorescence
It contains the design principle, working mechanism and bio- lifetime. These observed changes can be attributed to the heavy
logical application of the corresponding chemosensors. Further- atom effect (for Rb+ and Cs+) and/or a complexation induced
more, the design principles for the construction of selective change in triplet energy relative to the ground and excited
chemosensors for specific analytes and the problems and chal- singlet state energies as well as rigidification and conforma-
lenges encountered along the way will be discussed. We will end tional effects.
the review with a discussion of the future research directions and Potassium ions (K+) are one of the most important cations in
opportunities available for fluorescent chemosensors given that living organisms because they play essential roles in many bio-
they are now well-established research tools. logical processes. In mammals, the concentration of K+ inside
cells is about 150 mM, which is nearly 30 times higher than that
in the extracellular environment. Imbalances of potassium are
2. Fluorescent chemosensors for closely related to many diseases such as Alzheimer’s disease
cations (AD), anorexia, heart disease and diabetes. A number of fluor-
escent chemosensors for K+ have been developed. Currently,
There are a number of metal ions that play a vital role in our the 2-triazacryptand [2,2,3]-1-(2-methoxyethoxy)benzene (TAC)
daily physiological life. These include sodium (Na+) potassium group, which displays a very high selectivity for detecting
(K+), calcium (Ca2+), copper (Cu+ and Cu2+) and zinc (Zn2+), K+ over other physiologically relevant metal ions is the best
among others. However, some metal ions such as lead (Pb2+), K+-selective chelator. The TAC group was first reported by
cadmium (Cd2+) and mercury (Hg2+) are toxic and cause serious He et al. in 2003.9 They incorporated the TAC group into a
health and environmental problems. 4-aminonaphthalimide based polymer via an ethylene group,
in order to develop chemosensor 3 for the measurement of
2.1 Fluorescent chemosensors for alkali and alkaline earth extracellular (serum or whole blood) potassium based on a PET
metal ions mechanism (Fig. 1). The chemosensor rapidly and reversibly
As mentioned above, the first recorded fluorescent chemo- detects changes in potassium concentrations in whole blood
sensor for cations dates to 1867, when Goppelsröder reported samples. Furthermore, there were no interferences from clinical
that morin forms a strongly fluorescent chelate with Al3+. In the concentrations of Ca2+ or pH and from the interference of Na+
beginning, most fluorescent chemosensors for cations were even at concentrations of 160 mM. Additionally, this chemosen-
based on the coordination interactions between the hosts and sor has been used in the Roche OPTI CCA, a commercially
the guests. For example, Sousa et al. reported two naphthalene available whole blood analyzer, this system was developed in
based chemosensors 1 and 2 for the detection of alkali metal ions collaboration with de Silva (http://impact.ref.ac.uk/CaseStudies/
(Fig. 1). This resulted in the use of supramolecular chemistry in CaseStudy.aspx?Id=38360). Although TAC based chemosensors
fluorescent chemosensor design.8 Interestingly, these closely display high selectivities and sensitivities for K+, their syntheses
related chemosensors exhibit dichotomous behavior. 1 displayed often require lengthy synthetic routes and harsh reaction con-
a decrease in fluorescence quantum yield, also an increase ditions. Thus a significant demand exists for the development
in phosphorescence quantum yield, and a slight decrease in of more readily available ligands with the same properties
phosphorescence lifetime when it formed a complex with alkali displayed by TAC.
metal chloride salts in 95% ethanol glass at 77 K. On the The magnesium ion (Mg2+), which has a number of critical
contrary, complexation of 2 with potassium (K+), rubidium (Rb+), roles such as an enzyme cofactor, a DNA conformation stabilizer
or caesium (Cs+) chloride salts caused a noticeable increase in and a facilitator of transmembrane ion transport, is the most
fluorescence quantum yield, also a decrease in phosphorescence abundant divalent cation in cells. Abnormal concentrations of

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limitations of these two chemosensors are that they can only

be excited in the UV region, which hinders their development
for practical applications.
Calcium ions (Ca2+), are another important alkaline earth
metal ion. Ca2+ is the most abundant element in the human
body and it plays important roles in many biological processes.
The monitoring of the intracellular free Ca2+ is important since
imbalances of Ca2+ are related to a number of diseases such as
neurodegeneration, heart disease and skeletal muscle defects.
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The initially reported fluorescent chemosensors for Ca2+ suffered

several problems such as (1) low selectivity towards competing
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cations and in particularly H+ and Mg2+, (2) complex stoichio-

metry with Ca2+, (3) inflexibility of molecular design, or the
difficulty of rationally and iteratively adjusting chelator proper-
ties with different fluorophores.11 These problems were only
solved when Nobel Laureate Roger Y. Tsien developed two
Fig. 2 Structures of the fluorescent chemosensors 4–9.
1,2-bis(2-aminophenoxy) ethane-N,N,N0 ,N0 -tetraacetic acid (BAPTA)
based fluorescent chemosensors 6 and 7 for Ca2+ in 1980 (Fig. 2).11
Both of these chemosensors show good selectivity and sensitivity
Mg2+ ions are associated with many diseases such as migraines, towards Ca2+ over Mg2+, and their application in vivo opened
diabetes, hypertension and Parkinson’s disease. In order to up a new area and understanding of cellular function and
detect cellular magnesium ions, Farruggia et al. developed two indeed revolutionized our understanding of biochemical processes
8-hydroxyquinoline based fluorescent chemosensors 4 and 5 within cells.
for the detection of Mg2+ (Fig. 2).10 Initially, these two chemo- Along with the development of the field of fluorescence dyes
sensors show very weak fluorescence due to the an intermolecular and the advancement in microscopic imaging technologies, a
photoinduced proton transfer (PPT) and PET process. However, series of BAPTA and derived fluorescent chemosensors for Ca2+
after binding with Mg2+, the PPT and PET process are blocked, have been reported. In 2010, Kim, Cho and coworkers described
which results in a remarkable increase in the fluorescence a two photon (TP) fluorescent chemosensor 8, in which 2-(2 0 -
intensity. Chemosensors 4 and 5, with Kd of 44 and 73 mM, morpholino-20 -oxoethoxy)-N,N-bis(hydroxycarbonylmethyl) aniline
respectively, show high selectivity and sensitivity towards Mg2+ (MOBHA) was used as the Ca2+ receptor and 6-(benzo[d]oxazol-20 -
over other cations including Ca2+. These two chemosensors yl)-2-(N,N-dimethylamino)naphthalene was used as the fluoro-
have been used to image Mg2+ in live cells. However, the phore (Fig. 2).12 Chemosensor 8 shows high selectivity for

Fig. 3 Dual-channel TPEF images of HeLa cells co-incubated with 8 and 9 collected at (a) green channel: 390–450 nm (8), (b) red channel: 500–560 nm (9),
and (c) merged image of (a) and (b). TPEF images of a mice hippocampal slice co-stained with 8 and 9 collected at (d) green channel: 390–450 nm (8), (e) red
channel: 500–560 nm (9) at a depth of 100–200 mm at tenfold magnification and (f) a merged image of (d) and (e). Excitation wavelength: 780 nm. Scale bars:
30 mm (a and d). Reproduced from ref. 12 with the permission of John Wiley & Sons, Inc.

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Ca2+ and was pH-insensitive at biologically relevant pH. To 2.2 Fluorescent chemosensors for d-block metal ions
understand the Na+/Ca2+ exchange process, which is an impor- In contrast to the above-described fluorescent chemosensors
tant process vital to Ca2+ homeostasis, the Ca2+ chemosensor 8 for alkali and alkaline earth metal ions, which are all based on
and Na+ chemosensor 9 were applied to the simultaneously coordination interaction, some chemical reaction based fluo-
detection of Ca2+ and Na+ near the cell membrane of HeLa cells rescent chemosensors for transition metal ions have been
(Fig. 3). The HeLa cells labeled with 8 and 9 emitted bright developed since these metal ions can trigger specific reactions.
two-photon excited fluorescence (TPEF) in the green channel The strategies of using such reactions for sensing analytes has
emission (390–450 nm), corresponding to Ca2+ ions detected by significantly broadened the field of chemosensors.
8 (Fig. 3a) and red channel emission (500–560 nm), attributed
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Copper (Cu) is the third most abundant transition metal in

to Na+ ions detected by 9 (Fig. 3b), the merged images generated the human body, it is an essential transition metal in living
by two-photon excitation are given in Fig. 3c. These two chemo-
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organisms because it is involved in various physiological and

sensors have been applied to monitoring Na+/Ca2+ exchange in pathological processes. Loss of copper homeostasis is linked
live tissues at depths of over 100 mm (Fig. 3d–f). with diseases such as Menkes (copper deficiency), Wilson’s
The proton (H+) (or hydronium ion (H3O+)) is one of the (copper overload), Alzheimer’s disease, prion disorders, neuro-
most important charged species and has a crucial role in many degeneration and cancer.
physiological and pathological processes including receptor- In 1997, Czarnik and co-workers reported pioneering work
mediated signal transduction, ion transport, endocytosis, on a rhodamine-B derivative and its ring-opening reaction for
homeostasis, proliferation and apoptosis, multidrug resistance sensing copper ion (Cu2+).14 As shown in Fig. 5, the fluorescent
and muscle contraction. Mitochondria, an important organelle, chemosensor 11 can undergo a selective hydrolysis reaction
has a critical role in cellular metabolism such as energy with Cu2+ and yield fluorescent rhodamine B as a product. This
production, signaling, cellular differentiation, cell growth and work generated a great deal of attention for the ring-opening
death. The unique function of the mitochondria depends on processes of rhodamine derivatives for use as a fluorescent
the pH. Therefore, monitoring mitochondrial pH and in parti- chemosensors.
cular, changes related to mitophagy, may provide insights into Based on the same reaction, the Li group developed a NIR
mitochondrial function under physiological and pathological fluorescent chemosensor 12 for Cu2+ (Fig. 5).15 Undoubtedly, 12
conditions. Sessler, Kang, Kim and coworkers developed a shows high sensitivity and selectivity to Cu2+ over other related
mitochondria-immobilized fluorescent chemosensor 10 to metal ions. It is particularly noteworthy that this chemosensor
measure pH (Fig. 4), consisting of a piperazine-linked naphtha- exhibits unique single-photon frequency upconversion lumi-
limide as a fluorophore with a cationic triphenylphosphonium nescence (FUCL). Thus the product formed by the reaction of 12
as the mitochondrial targeting group, and a reactive benzyl with Cu2+ can be excited with both 670 nm and 808 nm light.
chloride subunit for mitochondrial fixation.13 The chemo- Due to the low background signal associated with NIR excita-
sensor is non-fluorescent in neutral form due to the PET tion (808 nm) and NIR emission (730 nm), it has an extremely
process. However, the PET process is inhibited at acidic pH low detection limit of 3.2 ppb in aqueous solution. This is much
and results in a fluorescence enhancement. 10 can be used for lower than that of the Stokes’ fluorescence methods for excita-
quantitative measurement of pH in mitochondria and real- tion at 670 nm where the calculated detection limit is around
time monitoring of mitophagy in cells. These results indicate 6.5 ppb. Significantly, this chemosensor has been applied for
that 10 has significant potential to be applied in biological the diagnosis of Wilson disease in live mice and therefore offers
systems, and how simple structural modifications of an estab- some promise for diagnostic sensing.
lished PET pH-sensor can open up new routes towards emerging Under physiological conditions, copper exists in its stable
biotechnologies. oxidized Cu2+ and reduced Cu+ states. Detection of Cu+ is just

Fig. 4 Structures of the fluorescent chemosensors 10 and the proposed mechanism for detection of pH in mitochondria.

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Fig. 7 Structure and proposed mechanism of 14 for detection of Zn2+.

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Zinc (Zn) is the second most abundant d-block metal in the

human body, which is often found as pools of mobile ions in
specific tissues of the body. Failure in the homeostasis of free zinc
ions is closely associated with neurological diseases and free zinc
ions (Zn2+) are also involved in apoptosis (programmed cell death).
The majority of small-molecule fluorescent chemosensors for
mobile zinc ions comprise of a fluorophore and a chelating unit
containing tertiary amines. However, acidic pH can interfere with
the detection of Zn2+ using these systems. To overcome these short-
comings, the Lippard group have developed a spirobenzopyran
based two-photon fluorescent chemosensor 14 for Zn2+ (Fig. 7).17
Chemosensor 14 can selectively detect Zn2+ in the presence of other
Fig. 5 Structures and proposed mechanism of 11 and 12 for detection related metal ions over a wide range of pH from 3 to 7. The chemo-
of Cu2+.
sensor has been applied to imaging exogenous Zn2+ in the lyso-
somes of HeLa cells, endogenous Zn2+ in insulin granules of MIN6
cells, and zinc-rich mossy fiber boutons in hippocampal tissue of
as important as the detection of Cu2+, however, many fewer mice. Furthermore, the relatively large two-photon absorption cross
fluorescent chemosensors have been developed for Cu+ than for section (d = 74 GM) and far-red emission makes it ideal for imaging
Cu2+. The fluorescent chemosensor 13 developed by the Chang zinc ions in tissue at depths of 4100 mm with greater contrast than
group consists of a tris[(2-pyridyl)-methyl]amine (TPA) as the existing visible-light fluorescent chemosensors.
binding and reaction site, and a bioluminescent D-luciferin as Mercury (Hg) is one of the most prevalent deadly toxins on
the reporter (Fig. 6).16 Compared with common fluorophore earth, which arises from many sources such as gold production,
based chemosensors, these bioluminescent reporter based coal plants, thermometers, barometers and mercury lamps. In the
sensing platforms have low background and high signal-to- past several decades, a huge number of fluorescent chemosensors
noise. Chemosensor 13 shows good sensitivity and selectivity have been developed for the detection of Hg2+. Pioneering work by
towards Cu+ over other related species except for free Co2+ Czarnik and coworkers on a desulfurisation reaction used the
(100 mM) that gives a modest response with the chemosensor. thiophilic character of Hg2+.18 Initially, the fluorescent chemo-
However, the concentration of Co2+ (100 mM) is not considered sensor 15 is non-fluorescent due to the PET process. The addition
physiologically relevant since most Co2+ is found tightly bound of Hg2+ induces an enhancement in fluorescence, whereas other
to proteins. Significantly, 13 has been used to image labile metal ions except for Ag+ caused no interference (Fig. 8).
copper pools mouse model of non-alcoholic fatty liver disease.
The results indicate that hepatic copper deficiency and altered
expression levels of copper homeostatic proteins accompany
glucose intolerance and weight gain.

Fig. 8 Structures and proposed mechanism of 15 and 16 for detection

Fig. 6 Structure and proposed mechanism of 13 for detection of Cu+. of Hg2+.

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Fig. 9 Structures of the fluorescent chemosensors 17–19.

Tae, Shin and coworkers developed a rhodamine-based fluo- Over the past several decades, there have been a number of
rescent chemosensor 16 for Hg2+, which takes advantage of the fluorescent chemosensors developed for the detection of anions,
known Hg2+-promoted formation of 1,3,4-oxadiazoles from thio- which have used host–guest interactions or chemical reactions.
semicarbazoles (Fig. 8).19 Chemosensor 16 shows high sensitivity
and selectivity over other metal ions including Ag+ and Pb2+, 3.1 Fluorescent chemosensors for anions based on host–guest
which can also promote the desulfurization reactions. The interaction
chemosensor has been used to detect exogenous Hg2+ uptake In 1994, Czarnik and co-workers reported an anthracene derived
in C2C12 cells and in zebrafish in real time as well as to image fluorescent chemosensor 20 for pyrophosphate (PPi) containing
accumulated Hg2+ in zebra fish organs. polyazaalkane groups (Fig. 10).21 20 shows good selectivity
Due to the close relationship between the two fields of towards PPi over other anions including phosphate (Pi), which
‘‘chemosensors’’ and ‘‘molecular logic’’, a number of fluorescent possesses a similar structure to that of PPi. The high selectivity of
chemosensors with two or more binding (reaction) sites have 20 towards PPi results from the two polyammonium arms that
been utilized to construct molecular logic gate. For example, are geometrically disposed for binding the six external oxygen
Akkaya and coworkers reported three styryl-Bodipy based mole- atoms of the pyrophosphate anion. It was not until the start of
cular logic gates 17, 18 and 19 using Hg2+, Zn2+ and (or) Ca2+ as this millennium that the development of fluorescent PET anion
inputs (Fig. 9).20 With these chemosensors, the dithiaazacrown
ligand is used as a Hg2+ binding site, di-2-picolylamine (DPA) is
used as a Zn2+ binding site while the aza-crown ligand is used as
a Ca2+ binding site in 19. Using Hg2+ and Zn2+ as inputs, the
emission signaling of 17 at 570 nm responds in accordance with
molecular logic gate AND function. For 18, the structure works as
an AND logic gate when the absorbance is recorded at 623 nm.
However, when the absorbance data is collected near the longer
wavelength peak, it responds in accordance with XOR logic.
Chemosensor 19 is a three-input AND logic gate using Hg2+,
Zn2+ and Ca2+ as inputs when the emission signaling is recorded
at 656 nm.

3. Fluorescent chemosensors for

anions
The development of anion selective chemosensors lagged
behind that of cation chemosensors, due to the strong hydra-
tion of anions. However, the field of anion sensing is now a
relatively mature science in line with the field of cation sensing.
This was driven by the important roles anions play in biological
and industrial processes as well as the need to produce new
methods of sensing anionic pollutants in the environment. Fig. 10 Structures of the fluorescent chemosensors 20–22.

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sensors that function on the bases of using charge neutral as a fluorophore and a urea binding group. Chemosensor 25
receptors was developed; this was achieved by several of the displays an interesting ‘‘on1–off–on2’’ fluorescence response
authors of this review in a concurrent manner. towards F (Fig. 12).25 Initially, 25 shows typical pyrene emission
The use of a Zn2+ complex as a binding site for PPi has been and maximum at 394 nm (on1) in MeCN. However, upon addition
found to be a particularly successful strategy due to the strong of F, the fluorescence decreases (off) due to an electron transfer
binding affinity between Zn2+ and PPi. In particular, the Hong process occurring in the locally excited complex and the con-
group have extensively explored this area of chemosensor version of locally excited complex to poorly emissive excited
development. A representative piece of their work, contains a tautomer. Interestingly, upon further addition of F, a yellow
naphthalene derivative based Zn2+ complex 21 as shown in fluorescence turns on, while a new emission band centered at
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Fig. 10.22 The Ka for PPi of 21 was calculated to be to be 500 nm (on2) appears, which can be ascribed to a charge-
2.9  108 M1, which means that 21 can detect PPi in water transfer emission by the deprotonated receptor; the F deproto-
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at nanomolar concentrations. Furthermore, 21 is able to detect nation phenomena having been observed by several researchers
less than 1 equivalent of PPi in the presence of a 50- to 250-fold in analogous systems. The authors demonstrated these optical
excess of ATP. This is the first example of a metal complex that features can be observed in other neutral receptors containing
can discriminate PPi from ATP in aqueous solution. N–H fragments, this may provide new strategies for the design of
Taking advantage of minimal photo-damage, deep tissue fluorescent sensors for anions.
penetration and high signal-to-noise contrast of NIR fluores- Recently, Gale and coworkers designed and synthesized a
cent chemosensors, Smith and coworkers developed a cyanine series of fluorescent anion transporters 26a–f consisting of a
based Zn2+ complex 22 for in vivo optical imaging of tumors and naphthalimide fluorophore with urea or thiourea receptors
cell death events (Fig. 10).23 Zinc complex 22 can selectively attached (Fig. 12).26 Interestingly, these transporters show two
accumulate in prostate and mammary tumors in two different distinct localization modes within cells. The aromatic substi-
xenograft animal models and it is now commercially available. tuted transporters localize within the cytoplasm and the less
This is an excellent example of a fluorescent chemosensor being lipophilic alkyl substituted transporters are over time localized in
applied for real-life practical applications. specific vesicles. Furthermore, the aromatic substituted com-
Recently, Sessler, Anslyn, and coworkers reported two anion pounds 26c–f all induce cytotoxicity in cancer cell lines, with 26f
induced supramolecular assemblies of expanded porphyrins 23 inducing apoptosis of A549 cells while alkyl substituted 26a and
and 24 (Fig. 11).24 Porphyrins 23 and 24 can form supra- 26b are non-toxic towards cancer cells. These results suggest that
molecular polymers with several diacids, which can be used the toxic effects can be ascribed to changes in ionic or pH
as chemosensors for both anions and organic solvents. The gradients across intracellular membranes rather than the plasma
solubility, colour, and fluorescence of the assemblies changes membrane. This research is particularly important since it offers
dramatically when they were treated with Lewis basic anions or exciting new applications for fluorescent chemosensors of anions.
polar solvents, which could be caused by a decrease in the
extent of aggregation. The authors have demonstrated that this 3.2 Fluorescent chemosensors for anions based on chemical
system can be used as a chemosensor for identifying certain reactions
salts and various solvents by solubility, fluorescence or visible As well as the host–guest interaction based fluorescent chemo-
colour change. sensors for the detection of anions discussed above, a number of
A urea and thiourea moiety can be used in the design of chemical reaction based fluorescent chemosensors for anions
various fluorescent chemosensors for H-bond donors. An excel- have been developed. These include the detection of reactive
lent example from the Fabbrizzi and Amendola group is the anions (reactive oxygen (ROS) and nitrogen (RNS) species). The
fluorescent chemosensor 25, which consists of a pyrene group superoxide radical (O2 ) is generated by the one-electron
reduction of molecular oxygen, which is the precursor of other
ROS and RNS. Thus, elucidating the relation between O2 fluxes
and diseases is of great importance. The group of Yang have
established a number of novel fluorescent chemosensors for
ROS/RNS. In 2015, they reported a series of fluorescein based
chemosensors 27a–c for O2 (Fig. 13).27 It is worth noting that
the trifluoromethyl group plays an important role in these
chemosensors. It is a strong electron-withdrawing group and
activates the sulfonate ester toward nucleophilic attack by O2 ,
yielding the free fluorophores. The trifluoromethyl group can
also prevent interference from cellular reductants such as
cysteine (Cys) and glutathione (GSH). All these three fluorescent
chemosensors can specifically detect O2 over other ROS/RNS
Fig. 11 Structures of fluorescent chemosensors 23, 24 and the schematic
and thiols. Furthermore, 27c contains a triphenylphosphonium
illustrates the construction of supramolecular assemblies using 23, 24, and group, which allows it to be used to monitor O2 changes in
diacids as the building blocks. mitochondria.

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Fig. 12 Structures of 25, 26a–f and the emission spectra taken upon addition of F to 25 (0.01 mM) in MeCN. Reproduced from ref. 25 with the
permission of the American Chemical Society.

signal transduction and antimicrobial activity, however, excessive

ONOO can damage critical cell components resulting in many
diseases. Recently, a boronate-based fluorescent chemosensor 29
has been developed for ONOO (Fig. 14).29 The chemosensor
displays relatively weak fluorescence due to the PET process.
A large fluorescence enhancement occurs upon the addition of
D-fructose. The interaction of the chemosensor with D-fructose
strengthens the fluorescence signal and in addition protects the
boronic acid from oxidation by other ROS/RNS. The system has
good selectivity towards ONOO over other ROS/RNS except
ClO due to its strong oxidizing ability. Additionally, the chemo-
sensor has been used to image endogenous or exogenous ONOO
in living cells.
Recently, the Qian group reported a FRET-based mitochondria-
specific fluorescent chemosensor 30 for the ratiometric detection
of ONOO (Fig. 14).30 The chemosensor consists of two cyanine
dyes (Cy3 and Cy5) and harnesses the differential reactivity of Cy3
and Cy5 toward ONOO. The chemosensor displays fluorescence
for Cy5 (660 nm) by FRET from Cy3 when excited at 530 nm.
However, upon addition of ONOO, a fluorescence increase at
560 nm and a decrease at 660 nm is observed which can be
Fig. 13 Structures and proposed mechanism of 27 and 28 for detection
ascribed to the selectively oxidation of the Cy5 moiety in 30 by
of O2 and ClO respectively.
ONOO. It is worth noting that both Cy3 and Cy5 moieties can be
oxidized by ClO. The chemosensor has been applied to imaging
Hypochlorite (ClO) is a prominent ROS, which plays an impor- both ONOO in live cells and the authors demonstrated that this
tant role in regulating invading microbes. However, uncontrolled fluorescent chemosensor can be used in semi-quantification of
production of ClO within phagocytes is acknowledged to be related cellular ONOO.
to the start of a number of human diseases. Yoon, Kim and Nicotinamide adenine dinucleotide (NADH) consists of one
coworkers described a two photon fluorescent chemosensor 28 adenine, one nicotinamide, two ribose rings, and a pair of
based on imidazoline-2-thiones (Fig. 13).28 Initially, 28 is non- bridging phosphate groups. Together with its oxidized form,
fluorescent in PBS buffer. However, upon addition of ClO, the NAD+, they are the most indispensable coenzymes and they play
fluorescence increases dramatically at 505 nm due to the formation important roles in multiple biological processes. Inspired by
of the corresponding imidazolium. Other ROS/RNS, do not induce the enzyme-catalyzed NADH sensing process, the group of
observable fluorescence changes, demonstrating outstanding selec- Chang have developed two resazurin based fluorescent chemo-
tivity of the chemosensor towards ClO. Chemosensor 28 also sensors 31 and 32 for NADH (Fig. 15).31 A two-step sensing
shows good sensitivity with a detection limit of 0.071 mM towards mechanism was proposed as shown in Fig. 15, first, the boronic
ClO and has been applied to imaging ClO in live cells and tissues. acid in 31 undergoes an esterification reaction with the diols of
Peroxynitrite (ONOO) is a strong oxidant observed in NADH. Second, reduction of the weakly fluorescent resazurin to
physiological and pathological processes. It plays a key role in the strongly fluorescent resorufin occurs. It is worth noting that

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Fig. 14 Structures and proposed mechanism of 29 and 30 for detection of ONOO.

the first step can facilitate the hydride transfer from NADH to fluorescent chemosensors for small neutral molecules over
31 and accelerate the reaction in the second step. However, 31 recent years.
can only work in basic conditions, which limits its applications
in biological systems. To address this problem, the boronic acid 4.1 Fluorescent chemosensors for reactive sulfur species
in 31 was replaced by 2-(hydroxymethyl)phenylboronic acid, (RSS)
yielding the chemosensor 32 which can work in pH 7.4 buffer Intracellular thiols such as cysteine (Cys), homocysteine (Hcy)
solution (Fig. 15).31 It can evaluate NADH both in vitro and in and glutathione (GSH) play key roles in biological systems.
live cells. Although this chemosensor still has some drawbacks Abnormal levels of these molecules have been linked to a
such as photoinstability and ease of wash-out, this work provides number of diseases, such as liver damage, leucocyte loss, psoriasis,
inspiration by mimicking biological processes for the design of cancer and AIDS. Accordingly, the detection of these thiol-
fluorescent chemosensors. containing biomolecules in biological samples is very important.
While de Silva demonstrated the first use of PET sensors for
thiols in 1998,32a it was in 2004, Martı́nez-Máñez and coworkers
4. Fluorescent chemosensors for small developed two squaraine based fluorescent chemosensors 33a
neutral molecules and 33b for the detection of thiols (Fig. 16).32b Their solutions
showed colour changes from blue to colorless along with
Small neutral molecules such as reactive sulfur species (RSS) fluorescence quenching in the presence of thiol-containing
as well as some neutral ROS/RNS are essential for our survival compounds, which is attributed to the selective addition of
since they play a vital role in a range of physiological and thiols to the cyclobutene ring in the chemosensors. These are
pathological processes. Conversely, some small neutral mole- two representative examples of thiol chemosensors that cannot
cules like nitroaromatics (explosives), and nerve-gas are a distinguish Cys/Hcy and GSH.
threat to public health and safety. These, two important reasons The Guo group reported a pyronin B based fluorescent
have stimulated the development of a substantial number of chemosensor 34 for the discrimination of Cys/Hcy and GSH

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Fig. 15 Structures of 31, 32 and proposed mechanism for detection of NADH by 31.

using different emission channels (Fig. 16).33 Initially, free 34 is

non-fluorescent due to the PET process from the methoxythio-
phenol group to the pyronin moiety. Upon treatment of 34 with
GSH, a fluorescence enhancement at 622 nm occurs due to the
replacement of 4-methoxythiophenol moiety by the thiol group
of GSH. In the case of Cys/Hcy, an intramolecular rearrange-
ment occurs followed by a substitution reaction, which leads
to fluorescence enhancement at 546 nm. The chemosensor
has been applied to imaging Cys/Hcy and GSH in live cells.
Importantly, the use of the intramolecular rearrangement of
Cys/Hcy with chemosensors is a typical strategy for the design
of fluorescent chemosensors to discriminate Cys/Hcy and GSH.
More recently, Urano and co-workers reported two reversible
fluorescent chemosensors 35a and 35b for GSH (Fig. 17).34 These
two chemosensors show ratiometric fluorescence response to
GSH due to the FRET process. The Si-rhodamines were selected
as reaction sites because they achieve an intermolecular equili-
brium with GSH. As a donor fluorophore, the O-rhodamine was
selected owing to its excellent spectral overlap with the
Si-rhodamines. Upon addition of GSH, the emission of the
Si-rhodamines decreases while the emission of the O-rhodamine
increases. The fluorescence ratio of the O-rhodamine and
Si-rhodamine units resulted in Kd values of 0.6 and 3.0 mM,
respectively for 35a and 35b. Chemosensor 35b has been used
Fig. 16 Structures and proposed mechanism of 33 and 34 for detection to image and quantify GSH in live cells. The authors have
of thiols. shown that these fluorescent chemosensors are revolutionary

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Fig. 17 Structures and proposed mechanism of 35a and 35b for detection of GSH.
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tools for investigating how GSH dynamics are regulated in a discriminate two or more closely related species using different
physiological context. fluorescence channels.
Hydrogen sulfide (H2S) is the smallest member of the reactive Hydrogen selenide (H2Se) can be thought of as analogous to
sulfur species (RSS). It has been characterized as a crucial H2S and it has been shown to be involved in many physiological
gaseous transmitter. However, a variety of emerging data suggest and pathological processes. There are only a few fluorescent
that hydrogen polysulfide (H2Sn) might be the signaling mole- chemosensors for H2Se that have been reported to date. Recently,
cules instead of H2S. Given the importance of H2S and H2Sn in the Tang group reported a hemicyanine based NIR fluorescent
redox biology, the Xian group has developed several novel chemosensor 37 for H2Se, using the selective cleavage of Se–N in
fluorescent chemosensors for these two species. Recently, they benzoselenadizole by H2Se through nucleophilic displacement
prepared a fluorescent chemosensor 36, which enables dual- (Fig. 19).36 Initially, the free chemosensor is non-fluorescent due
channel discrimination between H2S and H2Sn (Fig. 18).35 The to the heavy atom effect of Se. However, after the addition of
design principle for this chemosensor is that H2S selectively H2Se, the chemosensor undergoes a ca. 10-fold ‘‘turn-on’’ fluores-
reacts with the azidocoumarin moiety, while H2Sn only reacts cence response. Additionally, it was successfully used for imaging
with phenyl 2-(benzoylthio)benzoate, which results in the endogenous H2Se in live cells and in mice.
corresponding fluorescence ‘‘turn-on’’. However, the real situa-
tion is more complicated since the azide group of 36 can be 4.2 Fluorescent chemosensors for other small neutral
partially reduced by H2Sn and the reaction of H2S with azides molecules
results in the formation of H2Sn (Fig. 18). However, due to Besides the anionic ROS/RNS, there are some neutral ROS/RNS
the FRET process, the reaction with H2Sn should just produce such as hydrogen peroxide (H2O2) and nitric oxide (NO), which
green fluorescence from rhodol. Furthermore, less than also play important roles in many biological processes. Lin and
0.5 equivalents of H2Sn are produced from the reaction of H2S coworkers reported a fluorescent chemosensor 38, which can
(1 equivalent) and azide, therefore the reaction with H2S can respond to H2O2, NO, and H2O2/NO with three different sets of
produce emission signals from both coumarin (major) and fluorescence signals (Fig. 20).37 Upon addition of H2O2, blue
rhodol (minor). Overall, 36 can detect H2S and H2Sn from distinct emission at 460 nm with excitation at 400 nm is observed.
emission channels. This chemosensor has been used to image However, when only NO is added, a rhodamine associated
H2S and H2Sn in live cells. Moreover, this work provides a enhancement in emission at 580 nm is observed when excited
strategy for developing fluorescent chemosensors that can at 550 nm. The chemosensor displays enhanced emission at

Fig. 18 Structure and proposed mechanism of 36 for detection of H2S and H2Sn.

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Fig. 19 Structure and proposed mechanism of 37 for detection of H2Se.

just 580 nm with excitation at both 400 nm and 550 nm in the

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presence of both H2O2 and NO due to a FRET process. Further-

more, it was shown to be capable of simultaneously monitoring
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endogenous H2O2 and NO in live cells.

Chemical explosives such as 1,3,5-trinitroperhydro-1,3,5-
triazine (RDX), picric acid (PA) and 2,4,6-trinitrotoluene (TNT)
are a threat to public health and safety. Therefore, fluorescent
chemosensors and sensor materials for the rapid and selective
detection of chemical explosives at trace levels are of great
importance. Anzenbacher Jr. and coworkers developed three Fig. 21 Structures of the fluorescent chemosensors 39a–c and 40.
pyrene based fluorescent chemosensors 39a, 39b and 39c for
RDX (Fig. 21).38 These chemosensors show ‘‘turn on’’ fluores-
cence response to RDX based on different mechanisms. The the detection limit towards PA was determined to be 26.3 ppb.
fluorescence enhancement of 39a can be ascribed to protonation All these results indicate that 40 is a proficient sensor material
of the tertiary amines upon deprotonation from RDX. While, the for rapid detection of phenolic–nitroaromatics. The authors
formation of iminium cation and imine can be the reason have previously demonstrated the use of Tröger’s base based
for fluorescence enhancement of 39b and 39c, respectively. naphthalimide structures as cellular imaging agents and as
Furthermore, these three chemosensors were used to construct potential anticancer agents.
a fluorescent assay to discriminate different analytes. Sulfur mustard (SM), a chemical warfare agent, is known to
Recently, the Gunnlaugsson group have reported a supra- be extremely toxic, quite stable, and easy to synthesize. Discovery
molecular Tröger’s base derived zinc coordination polymer 40 of fluorescent chemosensors for the selective and sensitive
for fluorescent sensing of phenolic–nitroaromatic explosives detection of SM is of great importance. In 2013, the Anslyn
(Fig. 21).39 The aqueous suspension of 40 displayed strong green group developed fluorescent turn-on chemosensor for a sulfur
fluorescence at 520 nm, due to the ICT transition. A selective mustard simulant 2-chloroethyl ethyl sulfide (CEES) based on a
fluorescence quenching was observed towards phenolic– metal-ion indicator displacement assay (IDA) (Fig. 22).40 In this
nitroaromatics (4-nitrophenol (4-NP), 2,4-dinitrophenol (2,4-DNP) system, they developed a supramolecular system containing two
and PA) in the presence of other competing nitroaromatic units including a receptor dithiol 41 and Cd2+–indicator complex
species. Furthermore, 40 displays reversible PA sensing and 42. Dithiol 41 can rapidly react with CEES to yield a podand 43,

Fig. 20 Structure and proposed mechanism of 38 for detection of H2O2 and NO.

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Fig. 24 Structures of the fluorescent chemosensors 45a–d.

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biomacromolecules often has huge consequences. The use of

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fluorescence imaging techniques provides a powerful tool for

studying these biomacromolecules and to fully understand their
purpose in these complex biological systems due to excellent
Fig. 22 Proposed sensing mechanism for the detection of CEES. spatial and temporal resolution and high molecular specificity.
The detection of biomacromolecules is a unique task as they
often have large molecular weights, complex structures and a
range of biological function. Over the past several decades, a
number of fluorescent chemosensors have been developed,
which have proven indispensable for bioimaging and the inves-
tigation of disease.
Czarnik carried out pioneering work on anthrylpolyamines
45a–d to sense polyanions such as heparin, poly-L-glutamate,
ds DNA (double-stranded DNA) and ss DNA (single-stranded
Fig. 23 Structure and proposed mechanism of 44 for detection of chiral
functional amines. DNA) in water (Fig. 24).42 These chemosensors display a red-
shift and decrease in their emission spectra when bound to
either ds DNA or to ss DNA. The chemosensor 45b is effective in
which exhibits high affinity to Cd2+ and hence displaces an indicator binding to polyglutamate while 45c an excellent binder of
(4-methylesculetin) from 42, leading to fluorescence enhancement at heparin. They have been used to monitor the activity of pronase
460 nm. The detection limit of CEES was found to be 0.2 mM, and and heparinase, respectively.
can detect nerve agent levels that pose a health risk. Schmuck and co-workers reported a pyrene-based peptide
Enantioselective fluorescent chemosensors is another hot topic beacon (fluorescent chemosensor 46) that was shown to inter-
in the field of chemosensors. It has gained tremendous interest in calate with DNA (Fig. 25).43 In solution, the folded conforma-
recent years. The Pu group have developed a series of enantio- tion of 46 exhibits a typical pyrene excimer emission. However,
selective fluorescent chemosensors for the recognition of some when bound to DNA the chemosensor undergoes a conforma-
important chiral compounds. In 2015, Pu, Yu and coworkers tional change to the unfolded form. The change in conforma-
reported a 1,10 -bi-2-naphthol (BINOL)-based bis(naphthylimine) tion leads to a ratiometric change in fluorescence from excimer
fluorescent chemosensor 44 for the detection of chiral functional (490 nm) to monomer emission (406 nm).
amines (Fig. 23).41 In the presence of Zn(OAc)2, 44 can react with For the purpose of protein labeling, Hamachi developed a
chiral amines to release 2-naphthylamine with a blue emission fluorescent semi-synthetic chemosensor 47 based on the ligand-
(l1 = 427 nm), which allows the substrate concentration to be directed tosyl (LDT) chemistry (Fig. 26).44 In this quencher-
determined. The combination of the remaining chiral binaphthyl tethered LDT (Q-LDT), the fluorophore (coumarin) is covalently
unit with the chiral substrates results in significant enantio- attached to a protein ligand and an azoic fluorescence quencher
selective fluorescence enhancements at l2 4 500 nm, which via a labile sulfonate linkage. When the ligand binds to a target
facilitates the determination of enantiomeric composition. Thus, protein surface, the sulfonate undergoes nucleophilic cleavage,
both concentration and enantiomeric composition can be deter- separating the coumarin fluorophore from the quencher. How-
mined by one measurement of the fluorescent response of 44. This ever, it still shows very weak fluorescence since the fluorophore
design principle represents a potentially general strategy for the and quencher remain close together within the ligand-binding
development of dual responsive fluorescent sensors. site of the protein. However, addition of exogenous ligand leads
to a fluorescence recovery as a result of the displacement of the
quencher-tethered ligand (Fig. 26). This chemosensor has been
5. Fluorescent chemosensors for applied for ligand binding assays of human carbonic anhydrase
biomacromolecules II (hCAII) and the SH2 domain in purified protein solutions as
well as in crude cell lysates.
Biomacromolecules are vital for the function of living bio- Recently, Kikuchi and coworkers developed a fluorescent
logical systems. However, the abnormal expression of these chemosensor 48 for heterochromatin protein 1 using the

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Fig. 25 Structures of the fluorescent chemosensors 46 and the schematic illustration of 46 and Its Interaction with nucleic acid (the photographs show
the corresponding cuvettes under UV light). Reproduced from ref. 43 with the permission of the American Chemical Society.

Fig. 26 Structures of the fluorescent chemosensors 47 and schematic illustration of the strategy for the Q-LDT-mediated construction of turn-on
fluorescent biosensors. Reproduced from ref. 44 with the permission of the American Chemical Society.

Fig. 27 Structures of the fluorescent chemosensors 48, anionic-48 and no-wash live cell imaging of protein labeling with 48 and maltose-binding
protein (MBP) (top) and MBP-PYP (bottom) expressed in HEK 293T cells. Reproduced from ref. 45 with the permission of the Royal Society of Chemistry.

photoactive yellow protein (PYP) as a tag (Fig. 27).45 The chemo- However, 48 can be rapidly digested by cellular esterases yielding
sensor consists of a hydroxy cinnamic as the PYP ligand, fluorescein the anionic-48 as shown in Fig. 27. Thus this chemosensor enables
the fluorophore, and nitrobenzene the quencher moiety. The no-wash selective labeling of intracellular proteins fused to the PYP
acetylated fluorescein was used in 48 because esters are membrane tag in a desirable time frame, without adhesion or accumulation of
permeable while digested fluorescein molecules are non-permeable. the tag or the probe with non-targeted organelles.

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Fig. 28 Structures of the fluorescent chemosensors 49a–e.

Alzheimer’s disease (AD) is a neurodegenerative disease that the need and importance of developing cancer biomarkers.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

has a massive effect on an individual’s memory, cognitive A particularly useful candidate for cancer imaging is cyclo-
abilities and personality.46 Amyloid-b (Ab) plaques are con- oxygenase-2 (COX-2), given that different levels are expressed in
Open Access Article. Published on 11 October 2017. Downloaded on 13/06/2018 07:29:12.

sidered be a key pathological biomarker for AD. Therefore, tumors and in inflammatory lesions.47 Peng and coworkers
the development of a chemosensor for the detection of Ab reported a fluorescent chemosensor 50, which can distinguish
plaques in vivo would be highly desirable for early diagnosis healthy cells from cancerous cells and more importantly can
and monitoring of AD. Ahn and co-workers have developed a distinguish cancerous cells from inflammatory cells (Fig. 29).47
number of two-photon dyes 49a–e as candidates for fluorescent In aqueous buffer, 50 is in a quenched folded-form, due to
chemosensors for Ab plaques (Fig. 28).46 The donor–acceptor the PET process. An ‘‘off–on’’ fluorescence response was
dyes developed were shown to be environmentally sensitive due observed for inflammations and cancers where COX-2 is over-
to the formation of intramolecular charge transfer (ICT) excited expressed. However the fluorescent emission is significantly
states. In hydrophobic environments such as organic solvents, different at the two sites due to different levels of COX-2 being
the dyes exhibited strong fluorescence. However, in highly polar expressed. For sites with inflammation, the fluorescence emission
solvents the probes were only weakly fluorescent. Therefore, (615 nm) increases gradually over a COX-2 range of 0–0.12 mg mL1.
it was believed that these chemosensors could be used for the While for sites with cancer the fluorescence emission (615 nm)
in vivo imaging of amyloid-b (Ab) plaques due to the cross-b decreases and a new emission appears at 555 nm over a COX-2
sheets of the amyloid plaques providing a hydrophobic environ- range from 0.12–3.32 mg mL1. Consequently, this chemo-
ment inside and a hydrophilic environment outside. Among those sensor has been used to develop a fluorescence protocol for the
NIR dyes, 49a was shown to be a novel fluorescent chemosensor selective discrimination of cancer over inflammation as shown
for the detection of Ab plaques. The chemosensor which pos- in Fig. 29.
sesses a considerable two-photon absorption cross-section value In contrast to fluorescent chemosensor 50, which is based on
at 1000 nm was shown to have the ability to penetrate the blood conformational changes, there have been several reaction-based
brain barrier (BBB) and allow in vivo imaging of Ab in a live fluorescent chemosensors for cancer using other biomarkers.
mouse model. b-Galactosidase (b-gal) is an exoglycosidase that catalyses
Over 7 million people die annually as the result of cancer, the hydrolysis of b-galactosides to generate monosaccharides
with the number set to rise over the next 20 years. This highlights through the cleavage of the glycosidic bond. b-Gal is widely

Fig. 29 Structures of the fluorescent chemosensors 50 and imaging tumors in vivo. (a) 50 (30 mM) was injected intravenously (30 mL). The incubation
time was 30 min. (b) Visualization of tumor resection by the naked eye under ultraviolet illumination. Reproduced from ref. 47 with the permission of the
American Chemical Society.

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Fig. 30 Structure and proposed mechanism of 51 for detection of b-gal.

Fig. 32 Structure and proposed mechanism of 53 for detection of ALP.
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recognized as a biomarker enzyme for cell senescence and

primary ovarian cancer. Zhu and coworkers recently reported have a high affinity for western blots. Furthermore, this design
strategy may deliver a general approach for the simple and rapid
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a ratiometric near-infrared (NIR) fluorescent chemosensor 51

for b-gal detection (Fig. 30).48 Upon hydrolysis, there was a detection of proteins using western blots.
visual colour change from a faint yellow to a rose red, which
allows the systems to be used for colorimetric detection. A 14-fold 6. Conclusions and outlook
fluorescence increase was observed in the ratio (I685nm/I500nm) or a
34-fold fluorescence increase was observed at 685 nm. This NIR The field of fluorescent chemosensors has developed signifi-
emission provided the opportunity for the chemosensor to be cantly over the 150 years since Goppelsröder reported the first
used for in situ and in vivo visualization of b-gal activity in fluorescent chemosensor for Al3+. In particular, we have
colorectal tumor mice models. The chemosensor was success- witnessed the explosive development of the field of fluorescent
fully applied for in vivo real-time capture of b-gal activity at a chemosensors over the past 50 years. The authors of this review
tumor site as visualized using high-resolution three-dimensional believe that this phenomenal growth can in part be attributed
imaging. to the pioneering research of Professor Anthony W. Czarnik’s
Recently, the Ma group have reported a cresyl violet based (DOB: 21-11-1957), and Professor A. Prasanna de Silva (DOB:
fluorescent chemosensor 52 for the detection of leucine 29-4-1952) who have inspired countless researchers through
aminopeptidase (LAP). LAP is known to be widely distributed their seminal contributions to the field of chemosensors and
in organisms from bacteria to humans, including various molecular logic. We prepared this tutorial review in order to pay
cancer cells (Fig. 31).49 The chemosensor shows a colorimetric homage to their pivotal contributions to this field and to wish
‘‘off–on’’ fluorescence response to LAP and the detection limit them both very happy birthdays in 2017. The review is also
was determined to be 0.42 ng mL1. Thus it can be used to important since it demonstrates how a field can develop and
monitor the concentration changes of trace amounts of LAP in flourish over a short 50-year period to become a recognized
different biosamples. The results indicate that cancer cells with and established branch of chemistry. This review highlights
a higher level of LAP show much stronger resistance toward representative examples of fluorescent chemosensors for
cisplatin. The authors demonstrated that LAP contributes to various analytes including cations, anions, small neutral mole-
intrinsic resistance and serves as a simple monitor to reflect the cules and biomacromolecules from around 40 groups. Readers,
relative resistance of cancer cells. requiring additional information are directed to the following
Alkaline phosphatase (ALP) belongs to a subfamily of phos- recent reviews.51–54
phatases, which are found in mammalian tissues. ALP is known On reading this tutorial review, it may seem to young
as a hydrolase enzyme, which is capable of catalyzing the hydro- researchers that all the great problems in chemosensors research
lysis of a phosphate ester from proteins, nucleic acids and other have already been solved. Nothing could be further from the
biological molecules. It has been suggested that elevated levels of truth, since we will always need ‘‘new’’ chemosensors for yet
ALP are linked to a number of diseases including cancer, unknown analytes. This could be in the form of new biomarkers
cardiovascular, bone and hepatic diseases.50 Nagano et al. devel- or trace pollutants in our air and water supplies. Also, biological
oped an ‘‘off–on’’ fluorescent chemosensor 53 for the analysis of and environmental analysis has increasingly stringent require-
western blots (Fig. 32).50 They achieved this through the attach- ments imposed by regulatory bodies, so while a current chemo-
ment of a phosphate group to the phenolic group of 2-Me-4-OMe sensor may work it may fall short of the required selectivity or
Tokyo green (TG), which became almost non-fluorescent. The sensitivity required for use in a specific practical application. So
hydrolysis of the phosphate ester by ALP resulted in a strong whether the problem requires bespoke new receptors or an
fluorescence enhancement and the chemosensor was shown to improvement of existing systems, we will continue to need an
increasing number of chemosensors to meet these challenges.
In summary, we expect that chemosensor research will continue
to expand and develop. As well as new and improved chemo-
sensors, we anticipate that new applications or approaches to
use existing fluorophores as chemosensors will emerge. For
example, Gunnlaugsson and Scanlan have repurposed a naphthal-
imide fluorophore in the form of a ‘‘pre-probe’’.55 These pre-probe
Fig. 31 Structure and proposed mechanism of 52 for detection of LAP. chemosensors consist of a targeting group (carbohydrate) which is

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