1 Introduction

Phosphates play a central role in the building of the most fundamental molecules in living organisms, such as DNA and RNA. Along with proteins and carbohydrates, DNA and RNA constitute the three major macromolecules essential for all known forms of life. Phosphates are also major constituents of membrane lipids (in the form of phospholipids) and are involved in many biological processes, including skeletal development and bone integrity, energy metabolism, cell sensing, and regulation of protein synthesis [1]. Approximately 85% of total body inorganic phosphate (Pi) is found in bone, primarily in association with calcium in hydroxyapatite crystals deposited onto the collagen matrix [2]. The remainder is in soft tissue, with only approximately 1% in extracellular fluids [3]. Prolonged Pi deficiency results in hypophosphatemia with accompanying serious biological consequences, such as impaired mineralization of bone resulting in osteomalacia or rickets, dysfunction of the central nervous system, increased erythrocyte membrane rigidity, abnormal function of leukocytes and platelets, weakness of rhabdomyolysis and muscle, and cardiac dysfunction and respiratory failure [4–7]. At the other end of the spectrum, hyperphosphatemia is now recognized to decrease life expectancy and lead to seizures, cardiac dysrhythmias, chronic kidney disease, muscle weakness and tetany, decreased visual acuity, soft tissue calcification, and eventually death [8–13]. Therefore, controlling the Pi concentration is critical for the well-being of the organism.

Inorganic pyrophosphate (PPi), the dimeric form of Pi and a by-product of cellular hydrolysis of ATP, DNA polymerization, and other metabolic processes, is a biologically important target given its role in many crucial reactions [14, 15]. The difference in PPi concentrations in a variety of biological environments may be a diagnostic marker for various clinical conditions. For example, abnormally high levels of PPi in synovial fluid are observed for patients with calcium pyrophosphate crystal deposition disease [16–19]. Hence, the sensing and imaging of PPi has become an important research target. Phosphates are not only essential factors in living organisms, they are also important as components of several medicinal drugs and fertilizers. Eutrophication in the aquatic ecosystem is often related to pollution from phosphates and phosphorylated compounds [20, 21]. Due to their importance in both biological and environmental fields, great efforts have been made to develop systems capable of selectively sensing phosphates and their related compounds. Several comprehensive reviews describing the recognition of phosphate molecules by artificial receptors have been published [22–32].

Molecular sensors for phosphate anions usually consist of a Pi/PPi binding (receptor) site covalently linked to a sensing unit, such as a fluorophore (Fig. 1). One of the main difficulties in designing appropriate binding sites for Pi arises from the high hydration energy of this anion (−2765 kJ/mol) that places it near the bottom of the Hofmeister selectivity series [33]. Moreover, most of the phosphate-type anions exist in water, at different pH, in different protonated states bearing different negative charges: H3PO4, H2PO4 −, HPO4 2−, and PO4 3−. For these reasons, most of receptors prepared in early studies work only in organic solvents, and even in such examples, it has been pointed out that the binding of the Pi/PPi anions to artificial receptors requires optimization of both electrostatic and hydrogen-bond interactions through topological complementarity. One of the strategies adopted in the design of receptors for Pi/PPi useful in aqueous solution involves the usage of easily protonated polycations, such as polyammonium, imidazolium, and guanidinium moieties. An early pioneer in this area was A.W. Czarnik, who designed and synthesized fluorescent chemosensors for PPi [34]. The sensing unit can be attached to the receptor (binding unit) directly so that the receptor is part of the conjugated π system of the fluorophore, or it can be separated from the receptor via a covalent linker (Fig. 1). Pi/PPi sensing is based upon a variety of signal transduction mechanisms, such as binding-induced modulation in the fluorescence/absorbance using a conventional molecular recognition means (e.g., hydrogen bonding and coordination chemistry). In recent years, more elaborate methods of fluorescence sensing have emerged which involve a metal displacement assay in which the metal ion in the complex is specifically removed by Pi/PPi, and the aggregation induces emission/quenching upon binding to Pi/PPi. Sensors exhibiting covalent bond formation/breaking upon coupling Pi/PPi binding have also been reported.

Fig. 1
figure 1

Schematic overview of fluorescence sensing using a molecular sensor for phosphate (P) consisting of a binding and a sensing unit. The sensor is shown as a “Turn-ON” fluorescent sensor, but “Turn-OFF” sensors are also plausible. The chemical structures of the representative binding and sensing units are displayed

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2 Molecular Sensors Based on Conventional Host–Guest Chemistry and Their Applications

Several interactions, such as electrostatic interaction, hydrogen bonds, metal ligand complexation or coordination chemistry, and hydrophobic interactions, have been employed for Pi/PPi binding, many of which are incorporated into the receptor site. From the viewpoint of the development of sensing systems in the last decade, the majority of the chemosensors for phosphate anions are rationally conjugated to an appropriate sensing unit, so that the binding events can be experimentally measured by spectroscopic changes resulting from excimer formation [35–39], intramolecular charge transfer processes (ICT) [40, 41], metal-to-ligand charge transfer processes (MLCT) [42] or photo-induced electron transfer processes (PET) [43, 44]. Receptor design still remains challenging, in particular for efficient sensing toward the complicated phosphate derivatives or the related biological events with high selectivity and sensitivity in aqueous solutions. Monitoring events over an appropriate time is also critical.

2.1 Hydrogen Bonding

The hydrogen bond is an attractive interaction between a hydrogen atom from a molecule or a molecular fragment X–H, in which X is more electronegative than H, and an atom or a group of atoms in the same or a different molecule, in which there is evidence of bond formation [45]. Common hydrogen bond donors generally used as a receptor for phosphates include amide, ammonium, imidazolium, guanidinium, pyridinium, and urea. The strength of the hydrogen bond is generally determined by electrostatic interaction, which is typically stronger than van der Waals force but weaker than the covalent or ionic bond. Therefore, most receptors contain multiple hydrogen bond donors that are carefully designed to provide a preorganized binding unit suitable for several different anions, including Pi/PPi (Fig. 2). Unfortunately, these receptors do not work well in aqueous solution, only showing an excellent sensing capability in pure organic solvent or an aqueous/organic solvent mixture because in organic solvent, the solvation energy is not that strong and supramolecular interactions play a more important role.

Fig. 2
figure 2

Schematic overview of the fluorescent sensors utilizing hydrogen bonding for specific binding with phosphates (P)

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2.1.1 Pi Sensing

Kumar and Srivastava [38] reported a protonated sensor built on a pyridine-2,6-bis-carboxamide framework as a binding unit and pyrene as fluorophore 1 (Table 1). This system showed a blue fluorescence corresponding to the pyrene monomer and a green fluorescence resulting from pyrene excimer upon the addition of NaH2PO4 or NaHSO4 in CH3CN. In the presence of perylene monoimide-based red emitter (PMI) and in response to the addition of these oxyanions, the sensor gave rise to white light emission visible to the human eye due to the panchromatic emission. UV/Vis and 1H-nuclear magnetic resonance (NMR) spectroscopy studies suggested the involvement of hydrogen bonding in addition to electrostatic interactions in the stabilization of the anion–sensor complex [38]. Because no evidence of energy transfer between the pyrene and PMI was found, the emission perceived as white light is simply a composite emission of the two fluorophores emitting independently.

Table 1 Inorganic phosphate sensors based on hydrogen bonding interactions
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A selective detection of H2PO4 − in CH3CN by a complete Turn-OFF fluorescence emission with tetraamide-based receptors bearing quinolyl moieties (2; Table 1) was investigated in 2013 by Kondo and Takai [46]. Similar but less significant fluorescence and UV–Vis changes of sensor 2 were recorded upon the addition of CH3COO−, HSO4 −, and Cl−. The results of the UV–Vis and fluorescence titrations of sensor 2 imply that the nitrogen atoms of the quinolyl groups play a crucial role in the discrimination between H2PO4 − and CH3COO− because they act as hydrogen bond acceptors for hydroxy groups of H2PO4 − while four amide NH groups act as hydrogen bond donors to recognize anionic oxygen atoms of H2PO4 − and CH3COO−. The high selectivity of H2PO4 − over CH3COO−was achieved because CH3COO−cannot form such hydrogen bonds due to the lack of a hydroxy group in CH3COO−. The fluorescence quenching induced by the association with H2PO4 − over CH3COO−could be attributed to PET.

A series of macrocyclic sensors based on benzimidazolium and urea appended with acridine 3 (Table 1) for ratiometric sensing of H2PO4 − were reported in 2013 by Martinez and Gao [47]. Adding 3.0 eq. of H2PO4 − tetrabutyl ammonium salt to the solution of sensor 3 resulted in quenching of fluorescence by 68% at 430 nm and enhancement of fluorescence by 4.3-fold at 501 nm, which could be attributed to the anion-induced acridine excimer, resulting in the fluorescent color change from blue (430 nm) to green (501 nm) (Fig. 3). However, HSO4 − was able to give rise to the excimer peak of the acridine derivative 3 at 501 nm. Ratiometric sensing seems to be a versatile principle for the class of benzimidazolium–urea-based receptors, showing that they could be used as ratiometric fluorescent sensors for H2PO4 − via the mechanism of anion-induced fluorophore dimer formation [47, 48].

Fig. 3
figure 3

Fluorescence titrations of 5 × 10−6 M sensor 3 (Table 1) excited at 357 nm with H2PO4 − in CH3CN. Insets: Top Normalized emission developments at the wavelength of monomer and excimer peaks as a function of H2PO4 − equivalents, bottom fluorescent color of sensor 3 in the absence or presence of H2PO4 − under a UV lamp excited at 365 nm. Reproduced from Zhang et al. [47] with permission from The Royal Society of Chemistry

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Luis, Vila, and coworkers [49] investigated an acridine-based pseudopeptidic receptor which showed a high fluorescence Turn-ON specific to H2PO4 − in CHCl3. The macrocyclic 4 (Table 1) was found to display an increase of fluorescence emission corresponding to a new band centered at 510 nm, whereas the original fluorescence at 420 nm disappeared in the presence of H3PO4. The response toward other anions was practicably negligible. As already mentioned, the solvent has an important effect on the recognition process. In water, the solvation energy is larger than the host–guest binding energy. Thus, spectra from titrations in water show an isosbestic point which results from an equilibrium observed between the triprotonated and the diprotonated receptor, and no supramolecular species are detected. In contrast, in chloroform, solvation energy is not as important, and supramolecular interactions play an important role. The behavior of compounds of 4 in acidic medium is comparable to the that shown by acridine in this study. Therefore, it can be concluded that the fluorescent moiety in the supramolecular complex formed between 4 and H2PO4 − in CHCl3 is always the acridinium fluorophore, which was confirmed experimentally via fluorescence pH titrations, fluorescence lifetime, 1H-NMR, and X-ray studies as well as by computational calculations.

A selective sensing of H2PO4 − in CH3CN driven by the assembly of anthryl amidopyridinium ligand 5 (Table 1) was demonstrated by Gong, Ning, and coworkers [50]. The ligand 5 showed a relatively low fluorescence intensity, which was ascribed to the quenching effect of a PET process from the anthracene moieties to the charged pyridinium ring. With an increase in the concentration of 5 in CH3CN from 10−5 to 10−4 M, a new excimer emission peak centered at 539 nm was observed, which might be due to the relative proximity of the anthracene moiety at higher concentrations. This result indicated that 5 has a tendency to aggregate to some extent at high concentration. Upon the addition of various inorganic anions, including Pi, to the ligand solution, the anthryl group of 5 exhibited a strong excimer emission via H2PO4 −-directed assembly, while other anions showed a negligible effect. The Job plot and elemental analysis confirmed the 1:1 stoichiometry for the 5-Pi complex. Considering there is only one anthracene fluorophore in the 5 structure and the appearance of the excimer emission between anthracene fluorophores upon addition of Pi, a plausible 2:2 stoichiometry between 5 and H2PO4 − was proposed, supported by absorption spectra and density functional theory (DFT) calculation [50].

2.1.2 PPi Sensing

Caltagirone et al. reported fluorescent symmetric bis-ureas [51] and asymmetric bis-urea receptor 6 [52], as shown in Table 2 for PPi recognition in dimethyl sulfoxide (DMSO) and in pure water once embedded in cationic surfactant cetyltrimethylammonium bromide (CTAB) micelles. A strong interaction between PPi and 6 was observed by 1H-NMR, and a 1:1 receptor/anion molar ratio of the adduct in the solid state was confirmed by crystallography of the analog of 6 and H2PPi2−. Upon addition of PPi, the decrease in the emission band of the naphthalene fragment at 376 nm was observed to be concurrent with the formation of a new emission band centered at 476 nm. The addition of other anions, such as Pi and F−, caused the formation of a new band at 476 nm, but its intensity was negligible compared to that observed in the case of PPi. The formation of a new red-shifted band can be attributed to a possible anion-assisted intermolecular π–π interaction involving the indole and the naphthalene groups, as suggested by theoretical calculations. Compound 6 is also able to selectively sense HPPi3− in pure water by means of fluorescence quenching once embedded in CTAB micelles.

Table 2 Inorganic pyrophosphate sensors based on hydrogen bonding interactions
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Molina et al. [53] reported a bis(carbazolyl)urea bearing two pyrene fluorophores (7; Table 2) as a selective receptor for the recognition of PPi in anhydrous CH3CN or CH3CN–H2O 85/15. In anhydrous CH3CN, 7 showed a broad and red-shifted emission band at 496 nm, assigned to the excimer emission of the pyrene moiety, and two sharp bands at 416 and 394 nm, arising from the monomer emission. The intensity ratio of excimer to monomer, I E/I M = 1.06, was barely changed in the concentration range of 10−7–10−5 M, indicating that the excimer emission results from an intramolecular excimer but not from an intermolecular interaction. Binding with PPi perturbs the intramolecular excimer emission of pyrene because the presence of the PPi interacting with the binding cavity forces the side chains to open in order to accommodate the guest into the cavity, thereby disabling any possibility of forming an intramolecular excimer. On the other hand, in aqueous mixture, 1H-NMR and quantum chemical calculations suggest that the 7·(H2O)(PPi)2 complex displays quasi-C2 symmetry and features all eight NH groups pointing inward to the cavity and an efficient pyrene−pyrene parallel stacking which induces an increase in excimer emission of the receptor upon binding to PPi.

2.2 Coordination Chemistry

While hydrogen bonding interactions have been widely utilized in the development of selective anion receptors and sensors in organic solvents [54], it has shown only limited success in selective anion recognition in aqueous systems [52, 53]. Because most phosphate anions and their derivatives are in water, it is crucial to bind and sense these species in aqueous medium. It is now regarded that one of the most powerful strategies for phosphate anion recognition and sensing in water is the utilization of coordination chemistry, where one or two vacant coordination sites of metal complexes are employed for binding anionic guest molecules [55–62]. In many cases, multiple metal ions are positioned on an organic scaffold at appropriate distances to allow an anion guest to bridge the metal centers, providing a means of introducing selectivity for a specific guest (Fig. 4).

Fig. 4
figure 4

Schematic overview of the fluorescent sensors utilizing coordination chemistry for the specific binding toward phosphate (P). M Metal

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While a number of metal ions have been used in such receptors, including those of the main group, transition metals, and lanthanides, Zn2+ is among the most commonly employed, particularly where the guests are phosphate derivatives [28]. Kimura and his group were the first to demonstrate that the macrocyclic Zn2+ complex exhibits excellent binding affinities to phosphate anions and the derivatives [63, 64], inspired by the binding sites of metalloenzymes, in which phosphates act as substrates or inhibitors by reversibly coordinating to one or more Zn2+ ions in the active site [65]. More recently, Hamachi and his group [43, 44] proposed the Zn2+-dipicolylamine (DPA) complex as a versatile binding motif for phosphate anions in water and biological fluids. The DPA is a tridentate ligand comprised of three nitrogen donors that provides good selectivity toward Zn2+ over other biologically relevant metal ions and leaves coordination sites free for Pi/PPi binding. One of the advantages of the Zn–DPA receptor is its simplicity in terms of synthesis and easy modification for optimization of sensor design. Subsequent to the studies of Hamachi et al. [43, 44], many types of Zn–DPA-based molecular/supramolecular sensors have been reported [28].

2.2.1 Pi Sensing

Bhalla, Kumar, and coworkers [55] reported the recognition of AMP and H2PO4 − in ethanol–tetrahydrofuran (THF; 3/1) using a Zn ensemble of a unique propeller-shaped hexaphenylbenzene derivative appended with quinoline moieties 8 (Table 3). By itself, sensor 8 does not exhibit any fluorescence emission in ethanol-THF (3/1) due to PET from imino nitrogen to a photoexcited hexaphenylbenzene moiety [66]. Upon addition of increasing amounts of Zn2+ ions (0–20 eq.) a strong fluorescence emission band appeared at 438 nm. This fluorescence emission band is attributed to the interaction between Zn2+ ions and imino nitrogens and the nitrogen atoms of the quinoline moieties as a result of which the PET is suppressed, resulting into fluorescence enhancement. Upon the addition of H2PO4 − between 0 and 6 eq. the emission band at 438 nm was drastically quenched, but continued addition of Pi to 6–12 eq. resulted in an increase in a new blue-shifted band at 366 nm. This result indicates the weakening of the existing 8–Zn bond due to the interaction of H2PO4 − with Zn. On the other hand, the addition of AMP led to the enhancement of emission intensity along with a slight blue shift in the signal from 438 to 431 nm while no significant fluorescence change was observed upon addition of other anions. A multichannel molecular keypad system based on three different chemical inputs (Zn2+, H2PO4 −, AMP) that switches between two different fluorescent outputs at 431 and 366 nm has been successfully constructed [55].

Table 3 Inorganic phosphate sensors based on coordination chemistry
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Yamato and collaborators [56] investigated a pyrenyl-linked triazole-modified homooxacalix [3] arene based ratiometric fluorescent receptors 9 selective for H2PO4 − ions (Table 3). This system exhibited a cascade signal output for the ligand toward Zn2+ and consequently H2PO4 − through switching of the excimer/monomer emission of pyrene at 485/396 nm from the “ON–OFF” to the “OFF–ON” type. The smaller downfield chemical shift of triazole proton suggests that the Zn2+ ion of complex 9 may be located in the negative cavity formed by the nitrogen-rich triazole ligand and the carbonyl group. In particular, the coordination force of the Zn2+ ion would prevent the three pyrene moieties from maintaining π–π stacking for the excimer emission and instead leads to a concomitant increase of the monomer emission of the pyrene in the fluorescence spectra. 1H-NMR results suggested that the receptor 9 and the H2PO4 − anion not only have strong coordination and electrostatic interactions but also have strong hydrogen bonding interactions. As a result, the ratiometric signal of I 485/I 396 of complex 9 changes from the OFF to the ON state upon addition of the H2PO4 − ion. A design of molecular logic gate using Zn2+ and H2PO4 − ions as the chemical inputs and the fluorescence emission at 396 and 485 nm as an output signal was also reported [56].

Ganjali et al. [59] reported a bis(8-hydroxy quinoline-5-sulphonate) cerium(III) complex 10–Ce as a novel fluorescence Turn-ON sensor specific for Pi recognition (Table 3). The significant fluorescence enhancement might be a result of the electrostatic interaction between Pi and the 10–Ce complex, in which two oxygen atoms of the Pi bridge interact with the center of Ce3+ to reduce the magnitude of the electron withdrawal via partial neutralization of the charge on the Ce3+ ion, thereby increasing the electron-donating character of the quinolone-5-sulphonate moiety and finally resulting in an increased efficiency of ICT [67].

The lanthanide (III) complex of bis-amine-substituted phenanthroline-based chiral ligand (11; Table 3) was subsequently synthesized and characterized in 2014 by Subramaniam, Mishra, Albrecht and their coworkers [60]. Although water is known to be a quencher of lanthanide complexes, both complexes show intense luminescence in HEPES buffer (10 mM, pH 7.4), which is attributable to a J = 2 transition (5 D 0 → 7 F 2) [68]. Complexes 11–Eu and 11–Tb show red and green emission, which displayed a selective sensing of Pi through 95 and 98% fluorescence quenching over other anions such as CH3COO−, NO3 −, and SO4 2−. A strong fluorescence quenching was also observed in the case of NO2 −, ATP, ADP, and AMP. The same selectivity was observed in the case of the bis-imine-substituted phenanthroline-based Eu complex reported previously [58]. Both complexes were shown to be useable as a cell-staining reagent to monitor phosphates present in biological membranes, as demonstrated in the case of the green microalga (Chlorella vulgaris CCNM 1017).

2.2.2 PPi Sensing

Kim and co-workers [69] reported a 1,8-naphthalimide–DPA–Zn (II) complex 12–Zn (Table 4) as a PPi-selective fluorescence Turn-OFF probe. The sensor 12–Zn exhibits significant fluorescence quenching upon binding to both PPi (approx. 52%) and ATP (approx. 31%) over other anions in 95% aqueous solution containing CH3CN. However, the binding of PPi induced a blue shift (approx. 23 nm), while a blue shift was not observed in the case of ATP. In contrast to the bis[DPA–Zn(II)] complexes discussed in other reports [70–72], where binding to PPi generally involved two Zn(II) ions, time-dependent DFT calculations at the level of B3LYP/6-31G (d) suggests that only one of the DPA–Zn(II) centers in 12–Zn binds directly to PPi. However, no experimental data to support the proposed binding mode between 12–Zn–PPi complex was provided in the study [69]. Molecular orbital studies predict that available electron density in either of the chelating moieties may participate in PET, which causes fluorescence quenching in the ligand. Binding of Zn2+ with chelating moieties eliminates the available electron density from the chelating moieties in 12 and, therefore, PET is not possible in the 12–Zn complex. Upon binding with PPi, the photo-induced charge transfer occurs from negatively charged PPi to either the DPA unit or a fluorophore moiety. A biological application was also attempted, namely, to monitor intracellular PPi by addition of 12–Zn to C2C12 cells with exogenous PPi.

Table 4 Inorganic pyrophosphate sensors based on coordination chemistry
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Wu et al. [70] demonstrated a PPi selective BODIPY-based fluorescence probe 13–Zn complex (Table 4) and its imaging in a living RAW264.7 cell line. Fluorescence quenching with 10-nm blue shift was observed upon the addition of PPi, whereas the other anions, including nucleoside polyphosphate (NPP), caused only minor fluorescence quenching or even induced a slight increase in fluorescence emission in a water–methanol mixture. 1H-NMR spectroscopy and DFT calculation suggested that Zn2+ binding occurs mainly through the four nitrogens at the N,N-di(pyridin-2-ylmethyl)-ethane-1,2-diamine substituent and confirmed the binding of PPi with the 13–Zn complex. These observations indicate that PPi binding to the Zn2+ results in weaker binding between 13 and Zn2+. Living cell imaging using the RAW264.7 cell line showed that the green fluorescence of the BODIPY-based 13–Zn complex disappeared when 1 eq. of PPi was applied.

The Zn complex of 1,1′-bi-2-naphthol bearing DPA units 14 (Table 4) that make PPi visible was investigated by Li, Yu, and their coworkers [71]. A fluorescence enhancement of 14 at 383 nm in 1% methanol/HEPES buffer was observed upon the addition of Zn2+ due to the suppression of PET from the lone pair of electrons on the DPA group. The fluorescence of the 14–Zn complex was subsequently increased by approximately fourfold with the addition of PPi, wherein the intensity was saturated at 0.3 eq. Among the other anions tested, ATP was found to also enhance the fluorescence of 14–Zn, and the selectivity coefficient for PPi and ATP was calculated to be 4.1/2.8, suggesting that the enhancement is dependent on both the bulkiness of the organic moiety and the number of phosphates. When the concentration of the 14–Zn complex was raised from 1.0 × 10−5 to 2.5 × 10−3 M it was possible to detect the recognition of anions by the naked eye through precipitation formation. The formation of small particles (0.8–1.3 μm), observed by scanning electron microscopy (SEM), indicated that 14 was uniformly dispersed in the solution, due to its poor solubility in the HEPES solution, but that upon the addition of Zn2+ to the system, the solution became clear and no small particles (turbidity) were observed. Once PPi was added, rough and scaly solids with large surfaces were observed which confirmed the interaction between the 14–Zn complex and PPi. The fluorescence imaging was demonstrated in HeLa cells treated with 14, followed by Zn2+/pyrithione and PPi.

König and Hamachi’s group [73] reported a rigid luminescent bis-Zn(II)-bis-cyclen complex 15–Zn specific for PPi (Table 4) in a completely aqueous solution. 15–Zn displayed a strong fluorescence quenching which could arise from a similar dimerization resulting in π–π stacking interactions of planar benzene–triazine moieties. Electrospray ionization mass spectrometry (ESI-MS) measurement of the complex in the presence of an excess amount of PPi showed that the major species for the complex corresponded to the receptor/PPi = 1:2 complex, and the presence of a dimer of the complex 15–Zn with PPi was clearly observed, enabling the isotope distribution to be compared with the predicted one.

Mateus, Delgado, and their coworkers [74] investigated a Zn complex of diethylenetriamine-derived macrocycle 16–Zn, bearing 2-methylquinoline arms and containing m-xylyl spacers (Table 4). At pH 7.4, the ligand exhibits almost no fluorescence emission. Single-crystal X-ray diffraction studies indicated that the ligand is bound to each Zn center by three nitrogen atoms of a diethylenetriamine subunit and a quinolyl nitrogen atom, and the metal coordination sphere is completed by the binding of oxygen atoms of carbonate anions. Upon the addition of 1 eq. of PPi to 16–Zn, a remarkable 21-fold enhancement of the fluorescence intensity at 368 nm was observed. The increase in fluorescence quantum yield of the complex can be attributed to a significant increase of the radiative decay constant of the complexed quinoline fluorophore. Further addition of PPi caused a decrease in fluorescence intensity, which may be ascribed to the removal of Zn2+ from the complex by the PPi anion. No further fluorescence change was observed in the presence of 1 eq. of the other anions studied, including Pi, NPPs, and phenyl phosphate, indicating that the 16–Zn receptor acts as a selective fluorescent sensor for PPi which leads to an important effect on excited-state intramolecular charge transfer (ESICT) in the quinoline ring. The selectivity of the 16–Zn complex is most likely related to a steric hindrance effect caused by the bulky quinoline pendant arms.

In 2014 a long wavelength detection of PPi using a simple terpyridine–Zn2+ complex 17–Zn (Table 4) was reported by Rissanen et al. [75]. The crystal structure of the 17–Zn complex confirmed the formation of the 1:1 metal complex in which Zn2+ adopts a distorted trigonal bipyramidal N3Cl2 coordination. The 17–Zn complex demonstrated an extraordinary sensitivity because the probe was able to detect PPi at nanomolar level with the lowest limit of detection of 0.8 nM, while the sensitivity of the other probes was invariably limited to micromolar levels. Although the unusual binding stoichiometry of 1:3 (PPi: 17–Zn) was confirmed unambiguously by a Job plot analysis and the binding constant of the 17–Zn and PPi was not available, a bright orange–yellow emission of 17–Zn–PPi was imaged in a single HeLa cell due to the excellent sensitivity of the receptor. Almost the entire cell could be mapped for PPi, even regions with minimal PPi concentrations (Fig. 5). The maximum emission intensities were recorded at the nuclei, and emissions were also observed from the membranous cytoplasmic structures. The receptor is highly suitable for monitoring the biological events occurring in a single cell.

Fig. 5
figure 5

Confocal fluorescence microscopy image of HeLa cells incubated with probe 17–Zn (50 μM). Reprinted with permission from Bhowmik et al. [75]. Copyright 2014 American Chemical Society

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3 Molecular Sensors Based on New Sensing Mechanisms and Their Applications

Recently, several alternative approaches to Pi/PPi sensing involving metal displacement, aggregation-induced emission or quenching, and chemical reactions have emerged as simple and convenient techniques with high sensitivity. In contrast to sensing based on supramolecular chemistry such as hydrogen bonding or coordination chemistry, when the maximum fluorescence change can be recorded with a time resolution in the order of seconds, the kinetics of reaction/displacement assay-based sensing strongly depends on the nature of reaction, which is several seconds in some cases [76] while in other cases it can be very slow and more than 1 h is required to reach the highest fluorescence change [77].

3.1 Displacement Assay

In this approach, a specific metal complex is designed such that Pi/PPi has a stronger affinity for the metal ion, resulting in displacement of the metal upon Pi/PPi binding which induces a change in fluorescence (Fig. 6). Most of the metal displacement sensors demonstrate a good reversibility because the addition of the metal ion can reverse the process of sensing. Another approach is the indicator displacement assay (IDA) in which an indicator is allowed to bind reversibly to a receptor in the first step. Pi/PPi with a strong affinity to the sensor is subsequently introduced into the system, causing the displacement of the indicator from the receptor, which in turn modulates the sensing unit (Fig. 6). IDA has become a popular method for converting almost any synthetic receptor into an optical sensor along the lines originally developed by Anslyn et al. [78, 79].

Fig. 6
figure 6

Schematic overview of fluorescent sensors utilizing the metal and indicator displacement assay for the specific binding toward phosphate (P). M Metal

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3.1.1 Pi Sensing

Meng, Zhang and their co-workers [80] investigated the fluorescein bearing 2-[(pyridin-2-yl-imino)methyl]phenol moiety 18–Fe complex (Table 5) as a receptor for the highly selective detection of Pi in THF-HEPES buffer mixture. The fluorescence emission of the 18–Fe3+ complex was completely quenched, which could be ascribed to the paramagnetic quenching effect of Fe3+ and/or MLCT. The ligand 18 displayed a high affinity to Fe3+ (K a = 1.40 × 106 M−1) over Cu2+ and other metal ions. The specific interaction between Pi and the 18–Fe3+ complex led to the liberation of fluorophore 18, and thus the fluorescence was approximately 9.6-fold recovered when 18 eq. Pi was added. The 18–Fe3+ complex showed a negligible fluorescent response upon the addition of diverse anions but rather displayed reasonable performance (less than fourfold) with other polyphosphate species, including NPP and PPi. Studies on the quantitative deposition of 18 in MDA-MB-231 cells and its fluorescence “ON–OFF–ON” response in living cells were conducted using a flow cytometer. Staining of the Pi-pretreated MDA-MB-231 and U-343 MGa cells with the 18–Fe3+ complex resulted in bright intracellular fluorescence images showing that 18 is able to display a fluorescence Turn-ON response to Pi in the living cells.

Table 5 Displacement assays specific to inorganic phosphate
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Another report of metal displacement assay in CH3OH was demonstrated by Hu, Ju, and their coworkers [81] in 2015. A steroid-coumarin conjugate 19 (Table 5) displayed a significant fluorescence quenching only in the presence of 9 eq. Cu2+ via PET resulting from the excited coumarin fluorescence in the presence of Cu2+ ions [67]. Furthermore, a 1:2 binding complex between the 1,2,3-triazole motif of 19 and the Cu2+ ion was shown using UV–Vis titration and ESI MS. When 2.0 eq. H2PO4 − ion was added, the absorption curve showed a dramatic change and almost reversed to the state of free compound 19, suggesting the removal of Cu2+ ion from the complex by H2PO4 − anion. Conversely, there was no change in fluorescence in the presence of other anions, including F−, Cl−, Br−,I−, CH3COO−, NO3 −, and HSO4 −. The Turn-ON recognition ability in fluorescence for histidine was also observed as a result of the release of the free 19 after Cu2+ was removed from the complex to form a stable Cu–histidine complex due to the universal interaction between Cu2+ and amino acids. Its application as a molecular logic gate was studied with Cu2+ and H2PO4 − as input signals and the fluorescence intensity at 405 nm as output. The same research group also reported steroid–salen conjugate 20 for Zn ion recognition and cascade recognition for H2PO4 − in CH3OH, showing the logic gate properties of Zn2+ and H2PO4 − [82].

3.1.2 PPi Sensing

Li, Yu, and their coworkers [83] investigated a Cd(II)-terpyridine-based complex 21–Cd (Table 6) as a reversible ratiometric fluorescent probe for PPi detection in CH3CN solution. The ligand has strong fluorescence emission at 421 nm, and there was a new emission peak at 553 nm upon the addition of Zn2+ (I 553 /I 421 = 10) or Cd2+ (I 553 /I 421 = 30) that did not respond to Li+, Na+, K+, or Ca2+ in CH3CN. The red shift of the fluorescence emission of the ligand after coordination with Cd2+ can be ascribed to the electron-donating ability of the anthracene group, which causes ICT from the anthracene to the Cd(II)-coordinated terpyridine (see structure in Table 6). The 2:2 binding stoichiometry of the ligand and Cd2+ was verified by a single-crystal structure analysis and mass spectrometry. Upon the addition of PPi to the 21–Cd complex in CH3CN solution, the ratio of the I 431 /I 560 increased from 0.26 to 7.46, whereas the addition of ATP increased this ratio only from 0.26 to 2.66. The fluorescent titration, UV–Vis absorption, and high-resolution mass spectrometry results suggested that PPi could completely de-complex the 21–Cd and liberate the ligand. This ratiometric sensing behavior of red and blue shifts upon addition of Cd2+ followed by the addition of PPi in fluorescence emission was reversible over five successive cycles. Finally, Li et al. [83] demonstrated the confocal fluorescence imaging of 21–Cd complex with PPi in living RAW264.7 cells.

Table 6 Displacement assay specific to inorganic pyrophosphate
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Zhu et al. [84] reported a near-infrared (NIR) fluorescent Turn-ON sensor 22–Cu complex, which employs dicyanomethylene-4H-chromene as the fluorophore and the iminodiacetic acid group as the receptor (Table 6), for the selective detection of PPi in aqueous solution. Since α-amino acid shows a good chelating property toward Cu2+ [85], the incorporation of a lithium iminodiacetate group in 22 has a double effect, namely, the coordination ligand and the improvement of water solubility. There was no significant change in the fluorescence emission in the presence of other metal ions—only the addition of 5 eq. Cu2+ resulted in an obvious decrease in fluorescence. Job plots and mass peak analysis revealed that 22 forms a 2:1 complex with Cu2+. Meanwhile, the fluorescence intensity in the NIR region of 675 nm was obviously enhanced and finally stabilized upon the addition of 15 eq. of PPi. The associated color change can be easily differentiated by the naked eye (Fig. 7). Here, the fluorescence can not be recovered to the original intensity of 22, which indirectly indicates that PPi does not form a sufficiently strong complex to completely remove Cu2+ from the 22–Cu complex. To test its usefulness in a biological context, the sensor 22–Cu was then applied to KB cells (human nasopharyngeal epidermal carcinoma cell) supplemented with 3 eq. PPi and monitored.

Fig. 7
figure 7

Photographic images observed of the 22–Cu complex (10−4 M) with the addition of inorganic pyrophosphate (PPi) at 5 eq. a Color change, b fluorescence emission change irradiated at 365 nm by a portable fluorescent lamp. Reproduced from Zhu et al. [84] with permission of The Royal Society of Chemistry

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A diformyl–quinoline-based reversible receptor 23 (Table 6) prepared by Ramesh, Das, and coworkers [86] exhibits selective fluorometric enhancement upon the addition of Zn2+ in methanol aqueous solution. It is possible that the weak fluorescence of 23 is a consequence of ICT and free rotation around the azomethine (C=N) carbon, which brings flexibility within the ligand. In the presence of Zn2+, 23–Zn forms a rigid phenoxo-bridged binuclear Zn complex which restricts the free rotation around the azomethine carbon, thus inhibiting the ICT process, which results in enhancement of fluorescence intensity. The formation of the binuclear phenoxo-bridged metal complex 23–Zn provides an anion binding site in each of the metal centers. The complete quenching of fluorescence was selectively observed toward PPi, while other monovalent anions and NPP showed negligible response. Job’s plot and ESI–MS data revealed a 1:1 binding stoichiometry between the 23–Zn complex and PPi, and the apparent association constant was determined to be 1.76 × 104 M−1. The change in the emission spectrum of the 23–Zn complex upon the addition of PPi can be understood by considering the strong binding affinity of PPi toward Zn2+, resulting in the release of free ligand in solution. Application of 23 as a molecular logic gate with two inputs (Zn2+, PPi) and one output (emission intensity at 480 nm), and the detection of DNA amplification by PCR assay were demonstrated. The estimation of bacterial cell numbers by indirect measurement of PPi generated from the PCR reaction of the bacterial 16s rRNA universal primer was also performed in vitro.

A dinuclear-copper(II) complex 24–Cu (Table 6) with two ammonium arms based on bis-2-[(pyridin-2-ylmethylamino)methyl]phenol as the coordinated unit was reported by Xie, Chen, and coworkers [87] in 2016. Using esculetine as a fluorescent indicator, IDA was carried out to obtain the high affinity with PPi over other anions in 10 mM HEPES buffer (pH 7.0). A fluorescence quenching of esculetine at 465 nm was observed upon the addition of 1 eq. of the 24–Cu complex. With the addition of PPi, the fluorescence of the solution containing 24–Cu–esculetine was recovered, indicating the liberation of esculetine from the ensemble. The ensemble of 24–Cu–esculetine was successfully applied to monitoring the hydrolysis reaction of PPi and the released PPi from the PCR process, exhibiting its potential application in enzyme activity screening and DNA sequencing.

A family of cyclic peptide receptors bearing the DPA–Zn complex highly selective to PPi was investigated by Butler and Jolliffe [88] using indicator displacement assays in water, saline solution, and Krebs buffer. All receptors strongly bound the coumarin-based indicator, but hexapeptide scaffold receptor 25–Zn (Table 6) had the highest affinity toward the indicator and PPi, with log K a values of 7.3 and 9.8, respectively. The DPA–Zn unit was capable of quenching the fluorescence of the coumarin sulfonate derivative [89, 90], and the addition of 1 eq. of PPi resulted in almost complete restoration of its fluorescence intensity while the addition of ATP or ADP resulted in fourfold and 3.5-fold fluorescence enhancements, respectively. On the other hand AMP, cAMP, phosphoserine, phosphotyrosine, Pi, and the polycarboxylates acetylglutamate and Ac-Glu-Gly-Glu were not able to displace the indicator from the receptor to an appreciable extent. The enhanced selectivity of the 25–Zn complex observed for PPi in the routinely used Krebs physiological buffer solution ensures the further development of 25–Zn complex in a biological assay for PPi.

3.2 Aggregation Induced Emission and Quenching

Some organic molecules that are almost nonfluorescent in solution, exhibit a high capability to emit a strong fluorescence upon aggregation (Fig. 8). This exciting fluorescence phenomenon was first noted by Tang et al. [91] in 2001 from a solution of 1-methyl-1,2,3,4,5-pentaphenylsilole. They referred to this phenomenon as aggregation-induced emission (AIE) and subsequently showed that the restriction of intramolecular rotation (RIR) in the aggregates was the main cause of AIE phenomenon. Unhindered intramolecular rotation of AIE molecules under the free state leads to efficient nonradiative decay of the corresponding excited states, making them nonemissive. In view of such interesting fluorescence behaviors, AIE phenomenon have been successfully utilized to design sensitive and selective chemosensors suitable for the detection of PPi in living cells.

Fig. 8
figure 8

Schematic overview of the sensors working in aggregation-induced emission and aggregation-induced quenching in combination with displacement assay upon binding with phosphate (P). M Metal

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Chao and Ni [92] reported a terpyridine–Zn complex 26–Zn (Table 7) for selective nanomolar PPi detection over ATP and ADP in DMSO aqueous mixture based on AIE and ICT. Complex 26–Zn bears a donor–acceptor (D–A) structure in which the carbazole group is the donor and the terpyridine–Zn part is the acceptor; therefore, an ICT effect was observed. In fact, terpyridine–Zn complexes bearing the D–A structure displayed different emission in solvents with various polarities, and especially weak emission was found in aqueous solution due to the strong polarity of water. When the terpyridine–Zn–PPi ensemble (3:1 binding mode) formed nanoaggregates with an average particle size of about 300 nm, as confirmed by dynamic light scattering (DLS), SEM and transmission electron microscopy, the quenching effect caused by a polar solvent, such as water, can be inhibited efficiently, leading to the strong emission at 515 nm, which is known as AIE. Other anions, including Pi, led to negligible changes for the emission of 26–Zn but NPP induced less effects on the emission than PPi. The sensor 26–Zn has been successfully employed for nucleus staining in living HeLa cells (Fig. 9).

Table 7 Aggregation induced emission/quenching specific to inorganic pyrophosphate
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Fig. 9
figure 9

Confocal fluorescence images of HeLa cells incubated with 26-Zn (5 × 10−6 M) for 30 min and then further incubated with 4′,6-diamidino-2-phenylindole (DAPI; 5 × 10−6 g mL−1) for 10 min. The blue channel for DAPI excited at 405 nm and the green channel for 26–Zn excited at 488 nm. Scale bar: 20 μm. This work is licensed under a Creative Commons Attribution 4.0 International License

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A tetraphenylethylene imidazolium macrocycle 27 (Table 7) was reported to be a selective fluorescence Turn-ON sensor of PPi by Zheng et al. [93] in 2015. This positively charged macrocycle showed a maximum AIE effect in DMSO aqueous mixture, in the presence of a half eq. of Zn2+ with 2.4 eq. of PPi, while other common inorganic anions gave almost no response. In the presence of Zn2+, the UV–Vis titration of 27 with PPi was very similar to that of 27 without Zn2+, indicating that the metal ions did not directly interact with the macrocycle but only with the PPi. The association constant of the complex, according to a 1:1 molar ratio between 27 and PPi, as confirmed by ESI MS, was calculated to be 1.41 × 104 M−1. The crystal structure of 27 indicates that the distance between the nitrogen atoms of each imidazolium unit was approximately 5.836 Å, while the longest distance between the oxygen atoms of each phosphate unit of PPi was 5.36 Å. Therefore, the cavity of 27 is composed of two imidazolium units and is suitable for the inclusion of one molecule of PPi to form a 27–PPi complex, driven by an electrostatic attraction. In the presence of Zn2+, the two component 27–PPi complex was transformed into a five component (27–PPi)4–Zn complex due to one Zn cation being coordinated by two 27–PPi complexes. With continued coordination, an aggregate of (27–PPi)4–Zn complexes formed, with the average diameter of aggregates being up to 2800 nm as confirmed by DLS; therefore, the strong fluorescence was probably due to a RIR mechanism [94].

Another AIE fluorescence mechanism using 5-chlorosalicylaldehyde azine 28 induced by Cu2+ (Table 7) followed by PPi was reported by Tong et al. [95] in 2015. The structure of salicylaldehyde azine derivatives provided the AIE characteristic, as well as the potential chelating sites for metal ions [96, 97]. Compound 28 showed a strong fluorescence in the DMSO volume fraction range of 10–50%, which was considered to be AIE fluorescence in its aggregated state. In contrast, in a high DMSO volume fraction range of 70–90%, a weak fluorescence was observed due to the free rotation of N–N single bond in solution state. According to DLS data, the mean diameter of the fluorescent aggregates of 28 was 533.5 nm. The addition of Cu2+ considerably reduced the size of the fluorescent aggregates to 279.4 nm, which might be due to the better solubility of the 28–Cu complex. The addition of PPi led to the generation of fluorescent aggregates once again, with a mean size of 506.0 nm, which was close to that of 28 in the same solvent system. The fluorescence response of ADP and ATP was 25% and nearly 100%, respectively, of that of PPi. Negative charges at neutral pH for these NPPs were supposed to be responsible for their coordinating ability with Cu2+. An application to the analysis of PPi in a protein removed from 50-fold diluted fetal bovine serum sample was also demonstrated.

On the other hand, a unique combination of displacement assay and aggregation-induced quenching (Fig. 8) was first introduced by Ojida and Wongkongkatep et al. [76] in 2014. The xanthene probe–Ce3+ complex 29–Ce (Table 7) was reported as a selective fluorescence sensor of PPi in methanol aqueous solution. The 29–Ce complex forms an aggregated polymer through bridging coordination interactions between 29 and Ce3+ ions; therefore the large fluorescence decrease of 29 upon complexation with Ce(NO3)3 is ascribed to self-quenching of the xanthene fluorophore in the aggregated state and the quenching effect of the coordinated Ce3+ ions. When PPi was added to a solution of the 29–Ce complex, its fluorescence increased drastically (I/I 0 = 318) upon addition of over 20 eq. PPi. ESI–MS showed that 29 mainly exists as the free ligand in the presence of 20 eq. PPi, indicating that coordination exchange occurs between 29–Ce and PPi to form a Ce3+–PPi complex and to liberate the fluorescent ligand 29. Other phosphate species, including NPP, and other oxoanions scarcely induced an increase in fluorescence except for Pi (I/I 0 = 28) and uridine-5′-triphosphate (I/I 0 = 30). The fluorescence detection of DNA polymerase-catalyzed nucleic acid amplification by the loop-mediated isothermal amplification method for viral infection diagnosis using the 29–Ce complex was demonstrated (Fig. 10).

Fig. 10
figure 10

a Mechanism of the fluorescent loop-mediated isothermal amplification (LAMP) assay using the 29–Ce complex. b Changes in fluorescence of the 29–Ce complex upon addition of the LAMP reaction solution. Each reaction was conducted in the presence of 0 (A), 2 (B), 20 (C), 200 (D), and 1000 (E) copies of white spot syndrome virus plasmid DNA. Conditions [29]: 5 × 10−6 M,  0.27 mM Ce(NO3)3, 25 mM MES (pH 6.8)–CH3OH (1:1), 25 °C, λex = 500 nm. The y-axis indicates the ratio of the fluorescence increase of each sample [(F − F c)/F o], where F, F c, and F o are the fluorescence intensity of the reaction sample, of the control reaction sample without plasmid, and of the solution of the 29–Ce3+ complex, respectively. c Photographs of solutions of the 29–Ce complex upon addition of LAMP reaction solutions. Adapted from Kittiloespaisan et al. [76] by permission of The Royal Society of Chemistry

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3.3 Chemical Reactions

Not only molecular recognition, but the binding interaction between host and guest molecules can be based on an irreversible formation of a covalent bond. Such indicators are described as a chemodosimeter [98]. Chemodosimeters require at least two functional units, namely, the reaction site, where the host binds to the analyte covalently, and the sensing unit, which is dependent on the interaction with the analyte. The analyte reacts with the sensor to create a new molecule with different optical properties (Fig. 11). Because of their high sensitivity and selectivity, the design, synthesis, and application of chemodosimeters in luminescence bioimaging of Pi/PPi have attracted increasing attention and become an active research field.

Fig. 11
figure 11

Schematic overview of the reaction-based sensing mechanism. M Metal, O covalently bound oxygen, P phosphate

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3.3.1 Pi Sensing

Zhou et al. [99] reported the fluorescence properties of compound 30 containing an oxalate moiety linked via an ester bond to the hydroxyl group of coumarin fluorophore. The selective reaction of 30 toward Pi led to the cleavage of the ester bond and liberation of the fluorophores (Table 8). This unique Pi-induced hydrolytic reaction was effective in DMSO–HEPES buffered solutions that produce a colorimetric change associated with a 62-nm red-shift in the UV–Vis absorption maximum and up to a 780-fold enhancement in the fluorescence intensity; in contrast the addition of uridine monophosphate (UMP) and guanosine monophosphate (GMP) to solutions of 30 resulted in minor fluorescence enhancement (UMP 5.3-fold, GMP 3.6-fold), while common other anions, including NPP, cysteine, glutathione, and glutamic acid did not promote any change in emission. DFT calculations depicted the energy changes occurring in the hydrolytic reaction to be about −19.3 kJ. Mass spectrometry analysis of a mixture of 30 and 100 eq. of Pi in DMSO–HEPES buffer after stirring for 12 h contained a peak at m/z 151.08 corresponding to the cyclic diphosphate (Table 8). Fluorescence imaging studies were carried out using HeLa cells and Caenorhabditis elegans with the addition of 4 eq. exogenous Pi, or ATP and apyrase. A clearly detectable bright blue fluorescence was emitted, indicating the increase in endogenous Pi as a result of ATP hydrolysis catalyzed by Apyrase. Another application was performed using Sf9 adherent cells derived from Spodoptera frugiperda pupal ovarian tissue and treated with innexin 2 or 3 as an apotosis inducer. Treatment of Sf9 cells with 30 and innexin 3 in the absence of Apyrase resulted in a clearly detectable fluorescent image, thereby demonstrating that innexin 3 caused dephosphorylation of Akt (protein kinase B) in hemichannel-closed cells that led to apoptosis.

Table 8 Sensing through reactions specific to inorganic phosphate/inorganic pyrophosphate
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3.3.2 PPi Sensing

Zelder et al. [100] reported a metal disassembly strategy concurrent with reaction for imaging endogenous PPi in the mitochondria of HeLa cells by using the square pyramidal Fe3+–salen complex 31 (Table 8). Non-fluorescent 31 displayed an approximately 36-fold increase in emission intensity upon the addition of PPi in aqueous solution (pH 7.4), with a saturation of the intensity at approximately 25 min. Minor increases in emission were observed following the addition of NPP and Pi, but no other anions showed any effects. UV/Vis spectroscopy suggested that the presence of PPi led to a disappearance of the MLCT band of 31. Job plot analysis indicated a 2:1 stoichiometry for the reaction between 31 and PPi at physiological pH, and an equilibrium constant value (log K) of 7.06 ± 04 was calculated. The reaction-based release of fluorescent salicylaldehyde upon disassembly of 31 in the presence of PPi was confirmed with ESI MS, and the fluorescence properties of salicylaldehyde were compared with the reaction mixture.

A reversible covalent bond destruction leading to a change in optical properties of the sensing unit upon specific binding to the target molecule is also possible (Fig. 11). This unique sensing mechanism was first noted by Ojida and Hamachi et al. [101] and was used for an excellent design of a Turn-ON fluorescent PPi/NPP sensor. Sensor 32 comprises two DPA–Zn moieties as a binding motif for the PPi and the xanthene fluorophore as a sensing unit (Table 8). In the absence of PPi/NPP, the bridging water between the two Zn2+ centers attacked the ring of xanthene fluorophore breaking down a fluorescent π-conjugated ring under a wide pH range of 6–9 in 100% aqueous solution. Upon molecular recognition of PPi/NPP by the two Zn centers, the coordination geometry was modulated as the attacking water was removed to recover the fluorescent xanthene conjugated ring (Fig. 12).

Fig. 12
figure 12

Schematic illustration of a combination between chemical reaction and coordination chemistry as a reversible destruction of the covalent bond between bridging oxygen and xanthene ring results in a unique sensing mechanism of inorganic inorganic pyrophosphate (PPi)/nucleoside polyphosphate achieved by compound 32

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It should be noted that this novel reversible mechanism is a result of the good cooperation between the chemical reaction and coordination chemistry which demonstrates a high potential for several bioanalytical applications, including fluorescent staining of intracellular ATP storage in live Jurkat cells [101], fluorescent assay of the hydrolysis pathway of diadenosine tetraphosphate [102], organelle-localized multicolor fluorescence probes useful for imaging of NPP dynamics in living cells (Fig. 13) [103], detection of nucleic acid amplification reaction useful for viral infection diagnosis [104], detection of pathogenic spore-forming bacteria through the intracellular ATP pool [105], and fluorescent visualization of red blood cell complement receptor 1-mediated ATP release [106].

Fig. 13
figure 13

Confocal fluorescence analysis of HeLa cells stained with green 32 bearing a biocompatible anchor for membrane unit and pink 32 with a positively charged pyronin ring. Reprinted with permission from Kurishita et al. [103]. Copyright 2012 American Chemical Society

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4 Conclusion

In this review we have summarized how small molecular sensors turn their fluorescence ON or OFF in response to organophosphates, including Pi and PPi as well as potential applications. Hydrogen bonding interaction is quite useful for sensing in organic solvent where the hydration energy is weaker than the self-assemble capability between the sensor and the target molecule. Coordination chemistry provides a great advantage for selective binding of an oxoanion in a complete aqueous solution or the mixture. Several new sensing mechanisms, such as displacement assay, aggregation-induced emission, and chemical reactions enable the high sensitivity and selectivity for Pi/PPi detection. Towards this goal, new molecular design and sensing mechanisms which realize specific sensing of each phosphate species, including Pi/PPi, are urgently required. The development of a new sensing chemistry applicable to practical use is a worthy challenge for many chemists.