Fluorogenic sensors capable of selective interaction with analyte, which leads to a change in the position or intensity of the fluorescence band, allow to detect ions or molecules in situ and in vivo and possess high sensitivity and efficiency. Currently, they are widely used in organic, biological, and medical chemistry and environmental sciences for express monitoring of the ionic composition of the medium. They represent a serious alternative to the bulky, expensive, non-transportable technical devices traditionally used for this purpose, such as atomic absorption, atomic emission, and XRF spectrometers. Polyfunctional sensors capable of independent detection of two or more kinds of “guests” from a multiple mixture of cations, anions, or molecules due to specific spectral responses via the same or different channels constitute a rapidly developing area of chemosensory science. This specific feature is associated with the presence of two or more coordination centers in their molecules, or the capability of one center to selectively respond to various analytes with individual spectral changes. Coumarin (2H-chromene-2-one) core is one of the most versatile frameworks for the design of fluorogenic polyfunctional chemosensors for multianalyte detection. In this chapter, we report on the review of sensing properties of this group of chemosensors based on functionalized coumarin derivatives, including their applications in bioimaging.
- polyfunctional chemosensor
- amino acids
Сhemosensor is a molecule of abiotic origin capable of selective interaction with analyte causing corresponding changes in the physical properties of the initial system (absorption spectra, fluorescence spectra, etc.) . If a change occurs in spectral characteristics, the chemosensor relates to an optical type. There are two main types of optical chemosensors according to their mechanisms of action: chromogenic and fluorogenic [2, 3, 4]. In the case of chromogenic chemosensors after binding of analyte, there occurs a change in the electronic absorption spectra of the initial compounds. If this change can be seen with the human eye, we are dealing with a “naked-eye” chemosensor. Fluorogenic chemosensors can change their fluorescence spectrum after the interaction of analyte with receptor. It is highly desirable that this process is also accompanied by a “naked-eye” effect—contrast change in the color of emission. Chromogenic and fluorogenic chemosensor systems are widely used in organic, biological, and medical chemistry and environmental sciences for monitoring cations and anions. They represent a real alternative to the bulky, expensive, non-transportable technical devices, such as atomic absorption, atomic emission, and XRF spectrometers, that are traditionally used for this purpose. Of special efficiency are fluorogenic sensors, which use fluorescence for detection of various analytes, allow measurements in situ and in vivo, and are distinguished by highest sensitivity and selectivity. Recently a new scientific area in chemosensorics arose, which is associated with the design of poly- and bifunctional sensors capable of independent detection of two or more kinds of ions-“guests” due to the specific spectral responses via the same or different channels [5, 6, 7, 8]. Using a single molecule possessing different reactions against multiple analytes is cost-effective and useful for practical applications.
Herein we report on the review of spectral, fluorescent, and sensing properties of new representatives of this group of chemosensors based on functionalized coumarin derivatives, including their applications in bioimaging.
Coumarins (derivatives of 2H-chromene-2-one) constitute a comprehensive group of extensively studied heterocyclic compounds in organic, physical, and medical chemistry [9, 10]. Some coumarins have been selected as privileged scaffolds for drug design [11, 12, 13], and a number of antitumor, antiproliferative, antioxidant, antifungal, anti-inflammatory, and antiviral agents have been obtained on their basis [14, 15, 16, 17, 18, 19]. As a rule, substituted coumarins possess fluorescence in the visible part of the spectrum, as well as other useful photophysical properties. They are widely used in laser dyes, light-emitting devices, and solar cells. In addition, 2H-chromene-2-one is considered one of the most versatile frameworks for design of fluorescent, chemo- and biosensor systems [20, 21, 22, 23].
There are several excellent reviews devoted entirely or partially to coumarin chemosensors [24, 25, 26, 27, 28], but polyfunctional coumarin-based sensors for multianalyte detection until now have not been considered.
2. Sensing of multiple metal cations
Fluorescent polyfunctional sensors for detection of metal cations must contain a metal chelating or binding fragment attached to a coumarin core capable of absorbing and emitting light. The formation of complexes with ions should cause a change in the electronic structure or molecular conformation, which should result in an increase or decrease in the emission intensity.
A fluorescent sensor 1 (Figure 1) demonstrates a high selectivity toward Al3+ and Zn2+ in the presence of many various metal cations. Aluminum is the third most common element in the earth’s crust. It is used in food additives and cookware, although its cations are highly toxic and may be associated with Parkinson’s and Alzheimer’s diseases, microcytic anemia, dialysis dementia, and osteomalacia. Zinc is the second most abundant d-metal cation, which plays a crucial role in gene transcription, regulation of metalloenzymes, and transmission of nerve signals. However, it has some toxicity and its excess in living cells can cause neurodegenerative disorders, epilepsy, and seizures.
A new emission band at 427 nm (an increase in intensity ~500 times) in the presence of Al3+ in ethanol-water mixture appears due to hydrolysis of imine 1. The detection limit (LOD) was calculated to be 3.7 × 10−6 M. Detection of Zn2+ leads to a substantial initial fluorescence intensity enhancement at 489 nm due to the inhibition of PET process (photoinduced electron transfer) .
Coumarin 2 exhibits a significant enhancement of fluorescence intensity upon detection of Zn2+ (535 nm, 270-fold) or Al3+ ions (518 nm, 230-fold) in MeOH/H2O mixture with LODs 3.75 × 10−8 and 1.14 × 10−8 M, respectively . It has been shown that the sensing mechanism is based on inhibition of ICT process (intramolecular charge transfer) (Figure 2).
Compound 2 possesses a low toxicity and was used for fluorescent bioimaging of Zn2+ and Al3+ cations in PC12 cells.
Coumarin-crown compound 3 exhibits a high selectivity for the detection of Al3+, Cu2+, and Mg2+ in ethanol  (Figure 3). Copper(II) cations play an important role in biological systems. Their lack can lead to anemia and low white cell amount while an excess is accountable for neurodegenerative, Alzheimer’s, and Wilson’s diseases. Magnesium(II) cations are among the most abundant divalent cations in living cells. They are responsible for the formation of bone tissue, enzymatic biochemical reactions, cell proliferation, and DNA conformation stabilization, whereas their excessive concentration in the cytosol can lead to diabetes, hypertension, epilepsy, and Alzheimer’s disease.
While copper(II) is identified by color change of solution from a slight yellow to orange, Al3+ and Mg2+ ions cause a significant fluorescence enhancement at 592 nm and 547 nm with low detection limits of 0.31 μM and 0.23 μM, respectively.
Chemosensor 4 was developed for dual detection of Fe3+ and Zn2+ ions  (Figure 4). Iron(III) is the most common d-metal cation in the human organism, it plays a significant role in many enzymatic reactions and in specialized transport and storage of proteins. Its lack can cause anemia, diabetes, hemochromatosis, and Parkinson’s disease. Further development of this work led to the obtaining of a polyfunctional sensor 5 for Fe3+, Zn2+, and Cu2+ cations  (Figure 4). Formation of complex with Fe(III) ion and 4 or 5 is accompanied by a contrast color change from colorless to deep yellow.
Upon interaction with Zn2+ in CH3OH/H2O mixture, the emission intensity at 484 nm increases by five times compared to other metal ions. The LOD was found to be ~10−6 M. Since Cu2+ is a paramagnetic ion, its presence in the solution causes a substantial quenching of initial fluorescence of 4 and 5. The detection limit was calculated and found to be ~10−5 M.
A similar approach was used in design of a dual chemosensor 6  (Figure 5). While Fe3+ ions in EtOH/H2O solution cause only a visible color change from colorless to brown, the addition of Zn2+ ion resulted in 45-fold enhancement in the fluorescence intensity at 473 nm. The LOD was found to be 0.6 × 10−8 M.
Application of compound 6 as bioimaging fluorescent sensor for detection of Zn2+ in human cancer cells was also observed by fluorescent cell imager (Figure 6).
Chemosensor 7, as well as 4 and 5, in water (1% EtOH) solution demonstrates the quenching of the fluorescent properties when adding Cu(II) ions and significantly increases the emission intensity in the presence of Zn(II) ions  (Figure 7).
When Cu2+ and Zn2+ were monitored by sensor 7, the LODs were 141 nM and 72 nM, respectively. About 85% of cells survive upon addition of 80 μM of 7 indicating its hypotoxicity and possibility of using it for cell imaging (Figure 8).
Coumarin-naphthalene chemosensor 8 in the CH3OH/H2O mixture acts as a chemodosimeter for Ag+ ions and a fluorescent sensor in relation to Cu2+ ions . Silver is a potentially toxic and potentially carcinogenic element. It is known that Ag+ can react with proteins in the body, block thiol groups of enzyme systems, and inhibit tissue respiration. Excessive concentration of Ag+ ions in the body can lead to brain damage. Compound 8 possesses fluorescence at 480 nm, and its intensity is enhanced upon the addition of Cu2+ ions. However, the fluorescence intensity is quenched upon addition of Ag+ ions due to irreversible desulfurization (Figure 9).
The detection limits of 8 are 8.1 × 10−9 M and 44.0 × 10−9 M for Cu2+ and Ag+ ions, respectively. Compound 8 represents a safe and nontoxic to live cells biosensor. Only weak emission was observed in human osteosarcoma cells U-2 OS when exposed to 8, while strong blue and green fluorescence was observed upon addition of Cu2+ ions.
A dual-function coumarin chemosensor 9 could monitor Cu2+ and Hg2+ in CH3CN/H2O mixture . The addition of Cu(II) cations changes the solution color from yellowish-brown to yellowish-green, while Hg2+ causes the fluorescence intensity enhancement at 572 nm (Figure 10).
The detection limit for Hg2+ was calculated to be 2.96 × 10−7 M.
Coumarin chemosensor 10 for detection of Ag+ and Ce3+ ions in an aqueous solution was developed . Cerium is the most abundant of the lanthanides and refers to the conventionally toxic rare earth ultramicroelements. In the presence of Ag+, the color of the solution of 10 in EtOH/H2O changes from pale yellow to brown. The addition of Ce3+ ion results in the substantial emission intensity enhancement at 350 nm (Figure 11).
The detection limit of Ce3+ ion by the sensor 10 was estimated to be 2.07 × 10−7 M.
Selective fluorescent coumarin-triazole chemosensor 11 toward Ca2+ and Fe3+ ions was synthesized . Calcium(II) plays an important role in bone formation and as a second messenger in neurotransmitter release from neurons, in contraction of all muscle cell types and in fertilization. Inhibition of the PET process in 11 with Ca2+ ions leads to the fluorescence intensity increase at 473 nm, whereas complexation with Fe3+ causes its almost complete quenching (Figure 12).
The limit of detection was found to be 0.14 μM for Ca2+ and 0.25 μM for Fe3+.
3. Sensing of multiple anions
A very small number of fluorogenic polyfunctional coumarin-based chemosensors for multianalyte detection has been created so far. This is due to the fact that the recognition of anions is in principle a very difficult problem, since charges of anions are more diffused than those of cations, which leads to rather weak electrostatic interactions between anions and receptor part of the sensor. As a result, the receptors connected with the coumarin core must have the ability to either form of hydrogen bonds with anions up to complete deprotonation, or to nucleophilic addition reactions. Anions play an important role in medicine, biology, and industry. A deficiency of fluoride ions can cause gum disease and osteoporosis, and an excess leads to fluorosis due to its nephrotoxic action. Both excess and deficiency of bromide and iodide anions affect the functioning of the thyroid gland and can cause serious diseases. Acetate anion is involved in various metabolic processes. Cyanide ion is highly toxic to humans even in small concentrations due to its strong interaction with cytochrome-oxidase.
Coumarin chemosensor 12 is capable of detecting CN−, F−, and AcO− in the presence of other ions-competitors . Only cyanide anions caused a significant increase in the fluorescence intensity at 506 nm. However, only emission quenching was observed upon addition of CN−, F−, and AcO− to the solution of 12 in DMSO. According to 1H NMR titration data, this is due to the removal of the NH proton and the formation of the anionic form of 12 (Figure 13).
The fluorogenic and chromogenic chemosensor 13 in acetonitrile showed a change in the solution color upon addition of F− and AcO− ions from a yellow-green to red and orange, respectively . In an aqueous medium, 13 selectively reacted with cyanide anion via a nucleophilic addition reaction, and the nonfluorescent solution turned a fluorescent blue-green at 495 nm (Figure 14).
The addition of cyanide anions to an aqueous solution of 13 containing blood plasma caused a significant fuorescence enhancement at 450 nm by ~7.7 times and a change in the nonfluorescent color of solution to blue. The LOD to detect cyanide anion in blood plasma was found to be 0.37 mM.
Coumarin-thiazole chemosensor 14 for CN−, F−, and AcO− ions was synthesized . Compound 14 demonstrated a color change in DMSO upon addition of these anions from yellow to deep red in the visible region and yellow fluorescence with CN−.
1H NMR and DFT calculation data correspond to the deprotonation mechanism, while for CN− it is simultaneously supplemented by the addition reaction (Figure 15).
Coumarin 15 with the structure analogues to 14 was designed and synthesized for the selective detection of fluoride and cyanide anions . Chemosensor 15 in acetonitrile selectively reacts with F− via deprotonation mode, accompanied by a change in the color of the solution from colorless to deep red and a significant enhancement in the intensity of a yellow fluorescence. However, in an aqueous medium a substantial increase in the emission intensity at 506 nm was registered only in the presence of CN− ions (Figure 16).
The LOD of fluoride ions in organic medium is 0.72 μM, while for cyanide ions in aqueous environment the LOD is 2.7 μM.
Coumarin thiosemicarbazones 16 act via naked-eye and fluorescence mechanisms  (Figure 17). They were found to be selective chemosensors for F− with 1:1 receptor-anion ratio due to the appearance of a new emission band at 452 nm upon the addition of TBAF.
4. Sensing of metal cations and anions
As a rule, polyfunctional coumarin sensors for detection of metal cations and anions should include sites of various nature for the detection of these types of ions. Another displacement approach is based on the initial in situ formation of a complex with a cation, which then interacts with the anion, releasing the original sensor.
Diethylamine coumarin derivatives 17 and 18 were designed and synthesized to detect cyanide and copper(II) ions [45, 46]. The sensing mechanism is associated with the formation of a covalent bond between cyanide anions and 4-C carbon atom of coumarin (Figure 18). Red fluorescence of 17 at 669 nm is completely quenched, which is clearly visible to the naked eye. Complexation of 17 with Cu2+ exhibits color change from red to maroon and decreases the fluorescence intensity. LODs are 0.018 μM and 0.004 μM for CN− and Cu2+, respectively. Compound 18 also recognizes cyanide anions based on nucleophilic addition and copper(II) cations based on coordination reaction (Figure 18). However, in this case CN− causes an obvious enhancement of fluorescence at 473 nm.
The detection limits of the compound 18 are 0.0071 μM (CN−) and 0.014 μM (Cu2+).
Coumarin-based chemosensor 19 was obtained for selective fluorescent recognition of Cu2+ in MeOH/H2O mixture and subsequent detection of CN− via displacement approach . Compound 19 demonstrates strong emission at 448 nm, which is selectively quenched upon addition of Cu2+ due to the formation of the complex (Figure 19) with the LOD of 3.76 × 10−7 M.
This in situ prepared chelate easily reacts with CN− to form a very stable complex [Cu(CN)x]n− and the initial fluorescence is restored with the LOD of 1.35 × 10−6 M, which is much lower than the WHO limit of CN− (1.9 μM) for drinking water.
Coumarin 20 exhibits a significant increase in fluorescence intensity at 514 nm in the presence of Zn2+ ions, which is associated with the cessation of C=N isomerization process . The detection limit reached at 5.76 × 10−7 M. As expected, the Cu(II) cations almost completely quenched this fluorescence due to their inherent paramagnetic properties (Figure 20). The LOD reached at 3.1 × 10−7 M.
Thus, prepared in situ complex with copper regenerates the initial fluorescence of 20 upon addition of S2− even in the presence of F−, Cl−, Br−, and I− due to the formation of a very stable CuS. The LOD for S2− was measured to be 1.9 × 10−5 M.
Further development of this approach has been applied in the design of chemosensory systems 21 and 22 suitable for intracellular biology applications. Coumarin 21 demonstrates an intensive emission at 516 nm [49, 50]. The fluorescence intensity decreases (∼14-fold) upon addition of Cu2+ and reappears in the presence of S2− anions (Figure 21). The LOD toward Cu(II) was found to be 2 × 10−8 M, which is lower than the most of the values reported in literature, and toward S2−—6 × 10−8 M.
For the purpose of Cu2+ and S2− biovisualization, confocal fluorescent imaging was performed using A375 cells. It is clearly visible in the dark field images that green fluorescence is significantly quenched by Cu2+ and restored after subsequent treatment by S2− (Figure 22). The A375 cells were viable and maintained good shape in the entire process of this experiment, which means that 21 can successfully cross the cell membrane.
Similar results were obtained for 22. HeLa cells were incubated with 22 (10 mM) at 37°C for 30 min and displayed bright green fluorescence (Figure 23). After incubation with Cu2+ for another 30 min, the emission of cells decreased. Upon addition of S2− anions, the fluorescence intensity was restored. This indicates that 22 represents a potent candidate for sensing intracellular Cu2+ cations and S2− anions in living cells.
Upon addition of Hg2+ and F− ions, 23 underwent desulfurization and desilylation to induce an increase in the fluorescence intensity at 491 nm and 526 nm, respectively.
Aroylhydrazones 24 and bis-aroylhydrazones of coumarin 25 display the properties of bifunctional fluorescent and colorimetric naked-eye chemosensors for mercury(II) cations and fluoride anions detection [52, 53, 54] (Figure 25).
The addition of Hg2+ ions in acetonitrile solution of 25 allows to observe a distinct naked-eye effect with the change of color from pale-yellow to bright-orange. Simultaneously, the initial fluorescence is almost completely quenched. The LOD of mercury(II) cation sensing is 2.7 μM. In the presence of fluoride, cyanide, and acetate anions, a new absorption maximum in the visible spectral region appears. Furthermore, the formation of complex of 25 with fluoride anions is accompanied by the decrease in the relative intensity of the initial fluorescence I0/I in ≈22 times.
Coumarin-based chemosensors 26–28 with complex chemical architecture were designed and synthesized for selective sequential recognition of Cu2+ and pyrophosphate anion (PPi) [55, 56, 57]. PPi is the main product of adenosine triphosphate hydrolysis in living cells, which is involved in important metabolic processes. The structures of 26–28 and the sensing mechanism are shown in Figure 26. These compounds demonstrate on–off fluorescence quenching toward the Cu2+ cation due to the formation of complexes that show off–on fluorescence enhancement upon addition of PPi over many competitive anions.
Chemosensor 28 showed sequential on–off–on fluorescent bioimaging of Cu2+ and PPi in HeLa cells. After the addition of 28, the intense green fluorescence appeared (Figure 27). Cells incubated with Cu(II) cations efficiently quenched this emission, which was restored when treated with PPi. These data indicate that the sensor 28 possesses good cell permeability and can be used for bioimaging in live cells. The LOD for Cu2+ is 0.06 μM and for PPi it is 0.01 μM.
5. Sensing of metal cations and amino acids
Polyfunctional coumarin sensing of amino acids usually includes the initial detection of the appropriate metal cations, and in the second stage, the obtained in situ complex interacts with amino acid via displacement approach. A more complex problem is the creation of chemosensors capable of forming covalent bonds with the analyzed amino acid.
Amino acids are part of macromolecular proteins and represent essential substances for the growth and development of the human body. Cysteine (Cys) is of great importance in age defying, skin whitening, detoxifying, and improving immunity. Its deficiency causes premature senility, skin lesions, and uremia, while its excess can lead to senile dementia, neural tube defects, and osteoporosis. Histidine (His) is extremely important for the absorption of Fe2+ cations, vasodilation, and lowering blood pressure. The lack of His increases the risk of developing epilepsy, rheumatoid arthritis, and red cell aplasia, although its excessive content is associated with chronic kidney disease and Alzheimer’s disease. Arginine (Arg) plays a vital role in cell replication, wound healing, and protein synthesis.
A simple coumarin sensor 29 selectively detects Hg2+ and Cys in an aqueous solution  (Figure 28). The addition of Hg(II) leads to a hypsochromic shift of the fluorescence emission band, while Cys almost completely quenches the emission of 29. The latter process is seen by the naked eye under UV irradiation. The detection limit of 29 toward Cys is 8 μmol/L.
Coumarin 30 possesses a strong green emission at 527 nm . Upon the addition of Cu2+, 30 displays a significant fluorescence quenching. After the addition of Cys or His, the initial fluorescence is recovered due to the liberation of 30 (Figure 29).
Living A549 cells incubated with 30 exhibit notable emission. This fluorescence is quenched almost completely upon addition of Cu2+. Further incubation of cells with Cys and His leads to the restoration of the initial fluorescence.
With the addition of Cu2+, the solution of coumarin-rhodamine hybrid 31 in CH3CN▬H2O mixture exhibits a naked-eye change from pale yellow to pink  (Figure 30). The fluorescence color converses from blue to pink (new maxima appear at 490 and 615 nm, which correspond to the emission of coumarin and rhodamine B moieties, respectively). The LOD for Cu2+ is 0.47 mM. The 31-Cu2+ complex sequentially detects Arg with the restoration of blue fluorescence. The LOD for Arg is 0.33 mM.
The HeLa cells were incubated with 31 (20 mM) for 30 min, and a strong blue emission of the coumarin group was observed. Upon addition of Cu2+, the HeLa cells exhibit strong pink fluorescence. These data show that 31 is cell permeable and can be applied to fluorescence imaging of intracellular Cu2+ (Figure 31).
Coumarin 32 was prepared for the detection of Cu2+ and glutathione (γ-glutamylcysteinylglycine, GSH) . Overexpression of tumor biomarker GSH was found in many types of cancer. In the presence of Cu2, 32 exhibits selective fluorescence quenching and color change from yellow to orange-red. When GSH was added to the solution, the initial fluorescence was recovered (Figure 32).
The LODs were calculated as 2.40 × 10−8 M and 1.29 × 10−7 M for Cu2+ and GSH, respectively.
MCF-7 and HUVEC cells were both incubated with 32-Cu2+ complex for 30 min and then imaged under the same conditions (Figure 33). The fluorescence intensity in MCF-7 cells was above twofold higher than that in HUVEC cells, suggesting a higher GSH concentration in tumor cells. This is probably due to generation of additional GSH in tumor cells for resisting intrinsic oxidative stress.
Chemosensor 33 was designed for simultaneous detection of Cys, Hcy (homocysteine), and GSH . Due to different binding mechanisms, compound 33 demonstrates enhancing of fluorescence intensity with 108-, 128-, and 30-fold at 457, 559, and 529 nm for Cys, Hcy, and GSH, respectively, through different excitation wavelengths (Figure 34).
For exogenous biothiols, the BEL-7402 cells were firstly pretreated with NEM and cellular biothiols and SH-containing proteins were deactivated. After incubation with 33, no fluorescence could be observed (Figure 35).
After subsequent treatment with Cys, Hcy, and GSH, respectively, blue, red, and green fluorescence was observed from three different emission channels in living cells with high selectivity.
The design, synthesis, and investigation of fluorogenic polyfunctional coumarin chemosensors for multianalyte detection is an intriguing and extensively developing area of organic, medical, and biological chemistry. These sensors demonstrate high efficiency and selectivity combined with low cost and simplicity of analysis. Due to the limited size of the chapter, only sensors for the detection of metal cations, anions, and amino acids were considered, while sensors for proteins, DNA, RNA, etc. were ignored. Nevertheless, these data suggest that this group of polyfunctional chemosensors is extremely suitable for express analysis and bioimaging of various objects.
This research was financially supported by the Ministry of Science and Higher Education of the Russian Federation, project 0852-2020-2100-19. A. Dubonosov worked in the framework of the State assignment of the Southern Scientific Centre of the RAS No. 01201354239.
Conflict of interest
The authors declare “no conflict of interest.”