Recent advances in H₂PO₄⁻ fluorescent sensors

Abstract

Dihydrogen phosphate (H₂PO₄⁻) plays an essential role in a number of chemical and biological processes. The sensitive and selective detection of H₂PO₄⁻ is of great interest to many scientific fields, ranging from supramolecular chemistry to life sciences. For the detection of H₂PO₄⁻, fluorescent methods have plenty of distinct advantages, for example they are simplistic and allow low levels of determination. Therefore, this review will focus on the current progress in the development of H₂PO₄⁻ fluorescent sensors based on organic scaffolds, for sensing in both organic and aqueous solutions. Three main types of fluorescent probes will be categorized in this review: (i) intensity-based “turn-off” fluorescent sensors; (ii) intensity-based “turn-on” fluorescent sensors; and (iii) ratiometric fluorescent sensors that involve a ratio of two emission outputs. This review should provide a comprehensive description of this research area to date and be instructive for the design and synthesis of new fluorescent sensors for H₂PO₄⁻. In addition, the principles and mechanisms employed in the design of H₂PO₄⁻ sensors will be thoroughly described.

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Dawei Zhang

Dawei Zhang obtained his BS degree in chemistry from Northeastern University (NEU) in 2011, China. Subsequently, he started a co-sponsored PhD project between the École Normale Supérieure de Lyon (ENS-Lyon) in France and East China Normal University (ECNU) in China. He spent the first two years (2011–2013) in ECNU under the supervision of Prof. Gao. In September 2013, he moved to ENS-Lyon for three years supervised by Dr A. Martinez. His research interests are focused on anion recognition and fluorescent sensing as well as supramolecular catalysis.

James Robert Cochrane

James Robert Cochrane completed his PhD at the University of Sydney in 2011.Working with Katrina A. Jolliffe he studied the synthesis of cyclic peptide natural products. He then moved to the University of Melbourne, where he undertook a two year postdoctoral research fellowship with Craig A. Hutton. Here he worked on a number of projects including the synthesis of cyclic peptide hemicryptophanes for molecular recognition. After migrating to France, James is currently working as a postdoc in The Supramolecules, Materials and Stereochemistry team of the École Normale Supérieure de Lyon.

Alexandre Martinez

Alexandre Martinez received his Ph.D. in chemistry in 2004 from the University of Toulouse France, for studies asymmetric oxidation under the supervision of Dr B. Meunier. He did a postdoctoral work in 2004–2005 at the University of Geneva under the supervision of Prof. J. Lacour, working on supramolecular chemistry. He joined The Ecole Normale Superieure de Lyon in 2006 as associated professor, with Dr J.-P. Dutasta. His research activities include stereochemistry, catalysis, and supramolecular chemistry, in particular he focusses on hemicryptophane hosts.

Guohua Gao

Guohua Gao, Ph.D., Professor, received his Ph.D. in 1993 from Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences. He subsequently joined the Research Institute of Beijing Yanshan Petrochemical Corporation, SINOPEC. From May 1997 to December 2003, he worked respectively at National University of Singapore, Hokkaido University and University of Ottawa. Since August 2003, he has been a member of the Shanghai key laboratory of green chemistry and chemical processes, East China Normal University, China. His research interests are focused on imidazolium chemistry, with the special emphases on the catalysis by imidazolium based ionic liquids and the recognition by imidazolium based receptors.

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1. Introduction

Inorganic phosphate species are biologically relevant anions that have essential roles in genetic information storage, gene regulation, energy transduction, signalling processing and muscle contraction.^1,2 Phosphate is also a key constituent of two important biopolymers, DNA and RNA as well as many chemotherapeutic and antiviral drugs.^3,4 On the other hand, the over-use of inorganic phosphate in agriculture can lead to excessive algal growth, followed by decomposition and depletion of dissolved oxygen, and ultimately, the eutrophication of aquatic ecosystems.⁵ Dihydrogen phosphate (H₂PO₄⁻) is the predominant equilibrium species of inorganic phosphate at physiological pH. Therefore, H₂PO₄⁻ is an important target anion, and methods for its detection have received increasing attention of late.^6–10

The development of sensors for the recognition and detection of anions is of great importance in the field of modern supramolecular chemistry.^11–15 In this field fluorescent sensors bear inherent advantages, these include their high sensitivity, simple manipulation and facile visualization. All of these are essential properties for bio-imaging and thus the design of fluorescence-based probes for the detection of H₂PO₄⁻ is an important area of research.^16–26 Artificial fluorescent anion sensors belong to one of the following three design approaches (Scheme 1A): (a) the “binding site-signalling unit”, the interaction of the binding site(s) with anions, causes the change of the electronic properties of the signalling unit. Fluorescent mechanisms that commonly involved in this approach include photo-induced electron transfer (PET), the rigidity effect, fluorescence resonance energy transfer (FRET), excimer/exciplex formation/extinction, photo-induced charge transfer (PCT), and less frequently, excited-state proton transfer (ESPT). (b) The “displacement” protocol, in which the introduction of anions to the coordinated metal complex revives the non-coordinated spectroscopic properties of the indicator. (c) The reaction-based strategy which occurs between the target anions and the “chemodosisensor”. It is worth mentioning that until now, all the H₂PO₄⁻ fluorescent sensors have fallen into the first two categories, and there has been no reported chemodosisensor for H₂PO₄⁻ sensing.

Scheme 1 (A) The approaches for designing fluorescent sensors: (a) “binding site-signalling unit” approach; (b) displacement approach; (c) reaction-based chemodosisensor. (B) The possible fluorescent behaviours after anion binding: (i) “turn off” fluorescence; (ii) “turn on” fluorescence; (iii) ratiometric fluorescence. (C) The possible fluorescent mechanisms involved in the corresponding fluorescent behaviours.

The fluorescent phenomena observed after H₂PO₄⁻ binding also follows three main patterns of behaviour (Scheme 1B): fluorescence quenching, enhancement at the original wavelength, and ratiometric sensing that involves a comparison of intensities at two different emission outputs. Generally the fluorescent behaviour observed depends on the initial design of the sensor (Scheme 1C). (i) For sensors adopting the “binding site-signalling unit” approach, the mechanisms of PET and rigidity effect may quench or enhance the fluorescence of the probe after anion binding, while the FRET, excimer/exciplex formation/extinction, PCT, and ESPT can cause the bathochromic/hypochromatic-shift of the emission resulting in a ratiometric sensing behaviour. (ii) For sensors utilizing the “displacement” protocol, the fluorescent change of the coordinated complex induced by H₂PO₄⁻ is usually consistent with the initial non-coordinated indicator.

In the last decade, there have been reviews dealing with the subject of phosphate detection,^8,9 bio-phosphate recognition,^6,7,10 and pyrophosphate fluorescent sensing,²⁷ however to the best of our knowledge, there is no comprehensive review which thoroughly, systematically and timely describes fluorescent sensing of H₂PO₄⁻. As H₂PO₄⁻ is in equilibrium with two other basic anions HPO₄²⁻ and PO₄³⁻ at physiological pH,⁸ the selective sensing of H₂PO₄⁻ is especially important as well as challenging. For this reason in this review, we only highlight the detection of H₂PO₄⁻. In terms of the fluorescent behaviour observed upon H₂PO₄⁻ binding, we have classified the fluorescent probes based on their modes of action. Fluorescent probes exhibiting “turn-off” detection are covered in Section 2, “turn-on” in Section 3 and ratiometric sensing in Section 4. We subdivide the content of each section into the sensing mechanisms involved. This classification should promote a better understanding of the anion-induced fluorescent behaviour and will be instructive for the design of more selective sensors with the desired fluorescent properties. It should be made clear that in some cases, the mechanism of fluorescence is inferred by the authors and are not demonstrated absolutely. In some instances, two or more possible mechanisms might simultaneously exist in the sensing process, and in these situation, we place the sensor into the category where it can be best explained. It should also be highlighted that the selective recognition of H₂PO₄⁻ over other common anions is one of the most important issues to be addressed, thus the selectivity of each sensor has been evaluated.

2. Intensity-based “turn-off” fluorescent sensors for H₂PO₄⁻

In this section, fluorescent sensors that provide “turn-off” fluorescence detection of H₂PO₄⁻ will be described. Fluorescence quenching by H₂PO₄⁻ is a commonly encountered phenomenon which provides a facile approach for monitoring this important anion. PET is one of the most extensively adopted mechanisms for fluorescence quenching (Section 2.1 and Scheme 2). Upon the binding of H₂PO₄⁻, the PET process of the sensor is initiated or promoted (Scheme 2a), causing a corresponding decrease in the fluorescence of the sensor. The other methods used in fluorescence detection, such as “displacement” and ligand-to-metal charge transfer are discussed in Sections 2.2 and 2.3. For convenient comparison, the spectroscopic and analytical parameters of each “turn off” fluorescent sensor for H₂PO₄⁻ have been summarized in Table 1.

Scheme 2 Diagrams for (a) the initiation or promotion of PET and (b) the inhibition of PET after anion binding.

Table 1 Spectroscopic and analytical parameters of the “turn off” fluorescent sensors for H₂PO₄⁻

Sensor	Solvent	Fluorophore	λem (nm)	Fluorescent mechanism	H–G stoichiometry	Ka determined from fluorescence	Ref.
1	CH3CN	Anthracene	415	PET	1 : 1	1.3 × 10^6 M^−1	28
2	CH3CN–DMSO 9 : 1 (v/v)	Anthracene	415	PET	1 : 1	>1.3 × 10^6 M^−1	29
3	CH3CN	Anthracene	426	PET	1 : 1	1.6 × 10^5 M^−1	30
4	CH3CN	Anthracene	420	PET	1 : 1, 1 : 2	5.6 × 10^3 M^−1	32
5a	CH3CN–DMSO–H2O 98 : 1 : 1 (v/v/v)	Benzthiazole	452	PET	1 : 1	7.9 × 10^3 M^−1	33
6	CH3CN	Isoquinolyl	395	PET	1 : 1	2.5 × 10^6 M^−1	34
7	CH3CN	Quinolyl	350	PET	1 : 1	2.8 × 10^6 M^−1	34
8	H2O	Naphthalene	478	Displacement	1 : 1	1.8 × 10^6 M^−1	35
9	CH3OH–HEPES buffer 1 : 1 (v/v)	2,2′-Dihydroxyazobenzene	610	Displacement	1 : 1	1.6 × 10^4 M^−1	36
10	CH3OH	—	330	Ligand-to-metal charge transfer	1 : 2	1.0 × 10^5 M^−2	37

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2.1 Initiation or promotion of PET

The fluorescent sensor (1) reported by Kim et al.²⁸ (Fig. 1) bearing two imidazolium groups at the 1, 8-position of anthracene showed significant fluorescence quenching in CH₃CN upon addition of H₂PO₄⁻. This was due to the formation of (C–H)⁺⋯X⁻ hydrogen bonds. The binding constant of 1 with H₂PO₄⁻ was found to be relatively large (1.3 × 10⁶ M⁻¹), however strong competition by F⁻ for H₂PO₄⁻ binding was observed. To circumvent this, their group subsequently designed the rigid fluorescent sensor 2. Based on the scaffold of 1 two anthracene units were directly connected by the two imidazolium moieties forming a cyclic receptor.²⁹ Significant fluorescence quenching of sensor 2 occurred after addition of H₂PO₄⁻ in CH₃CN–DMSO (9 : 1, v/v) due to the PET process. More importantly, competitive binding studies demonstrated that there is no interference of the binding of H₂PO₄⁻ even when in the presence of 1.5 equiv. of F⁻. The binding constant of sensor 2 with H₂PO₄⁻ was also found to be larger than that of sensor 1 (>1.3 × 10⁶ M⁻¹).

Fig. 1 Structures of sensors 1 and 2.

Bearing the concept of the PET process between imidazolium binding sites and the excited state of anthracene in mind, Jadhav et al.³⁰ developed an anthracene-imidazolium-based macrocyclic sensor (3) (Fig. 2). In this sensor the two pendant imidazolium arms are connected by attachment to a molecule of cholestane.³¹ The sensor 3 exhibited 95% fluorescence quenching after addition of 10 equiv. of H₂PO₄⁻ in CH₃CN with a binding constant of 1.6 × 10⁵ M⁻¹, while only 20–40% decrease in intensity was induced by other common anions (F⁻, Cl⁻, Br⁻, I⁻, AcO⁻, HSO₄⁻). Competitive experiments demonstrated that the presence of excess anions (50 equiv.) did not cause any significant changes in the emission of 3 with H₂PO₄⁻ (5 equiv.).

Fig. 2 Structures of sensors 3 and 4.

Ghosh and co-workers have³² designed and synthesised a flexible anthracene linked benzimidazolium-based receptor (4) (Fig. 2) which could be used as a “turn-off” fluorescent sensor for H₂PO₄⁻ over other common anions. The fluorescence was quenched by 72% after addition of 2 equiv. of H₂PO₄⁻ in CH₃CN, while no other changes such as excimer emission were observed. The binding constant was found to be 5.6 × 10³ M⁻¹. A Stern–Volmer plot of sensor 4 with H₂PO₄⁻ indicated both static and dynamic quenching effects. Since further addition of H₂PO₄⁻ induced an increase in emission of sensor 4, the authors suggested a two-step process of complexation: 1 : 1 binding occurred initially, and then changed to a 1 : 2 (host–guest) stoichiometry when in the presence of excess H₂PO₄⁻.

A benzthiazole-based fluorescent sensor (5a) (Fig. 3) was developed for the detection of H₂PO₄⁻ by Lee and co-workers.³³ In order to evaluate its anion recognition performance, receptor 5b was synthesised. The results showed that among all the tested common anions, only H₂PO₄⁻ resulted in the significant fluorescence quenching of sensor 5a in CH₃CN–DMSO–H₂O (98 : 1 : 1, v/v/v) with a binding constant of 7.9 × 10³ M⁻¹, and no significant anion-binding interactions occurred with sensor 5b. The high selectivity of sensor 5a was attributed to the specific combination of both hydrogen bond-donating and -accepting moieties within the rigid cleft.

Fig. 3 Structures of sensors 5a and 5b.

Recently, Kondo et al.³⁴ reported selective detection of H₂PO₄⁻ in CH₃CN utilizing the fluorescence quenching effect. The synthesised tetraamide-based sensors (6 and 7) contain isoquinolyl and quinolyl moieties respectively (Fig. 4). Especially, sensor 7 bearing 2-quinolyl groups showed selective and nearly complete quenching by H₂PO₄⁻, whereas it showed small or no changes towards other anions. The high selectivity of sensors 6 and 7 for H₂PO₄⁻ (K_a = 2.5 × 10⁶ M⁻¹ and 2.8 × 10⁶ M⁻¹ respectively) can be attributed to the additional hydrogen bonds formed between H₂PO₄⁻ and the nitrogen atom of the isoquinolyl and quinolyl moieties.

Fig. 4 Structures of sensors 6 and 7.

2.2 “Displacement”: releasing the non-fluorescent indicator

A pyrimidine-naphthalene anchored Schiff base (8) was synthesised by Kumar et al.³⁵ which was used as a “turn-on” fluorescent sensor for Al³⁺ in aqueous solution due to the inhibition of C=N isomerization after Al³⁺ complexation (Fig. 5). The formed 8–Al³⁺ complex could also achieve the “turn-off” sensing of H₂PO₄⁻ with a detection limit of 2.27 × 10⁻⁷ M via protonation of aldimine-nitrogen of sensor 8 by H₂PO₄⁻. This releases the non-fluorescent 8 from the formed Al³⁺ complex. Unfortunately, HSO₄⁻ which has a lower pK_a value (1.99 vs. 3.88) also gave rise to the similar fluorescence quenching behaviour, while other common anions showed no fluorescent response.

Fig. 5 Sensing mechanism of sensor 8 towards H₂PO₄⁻.

The mononuclear 2,2′-dihydroxyazobenzene (DHAB)–Zn²⁺ complex (Fig. 6) was developed as a cost-effective “on-off” H₂PO₄⁻ sensor based on the “displacement” protocol in CH₃OH–HEPES buffer (1 : 1, v/v).³⁶ Obvious fluorescence quenching of the Zn²⁺–9 complex, after addition of H₂PO₄⁻ was observed due to H₂PO₄⁻-induced decomposition of the DHAB–Zn(II) complex releasing the non-fluorescent DHAB. An opposite increase in fluorescence was induced by CN⁻, while minimal or no changes were observed for other anions. The Zn²⁺–9 complex could also be used as a colorimetric receptor for H₂PO₄⁻ due to the marked solution colour changes.

Fig. 6 Sensing mechanism of sensor 9 towards H₂PO₄⁻.

2.3 Other mechanisms

Chen et al.³⁷ prepared a fluorescent tetranuclear pentacoordinated Zn(II) complex (10) based on a cresolic oxygen bridging ligand (L) shown in Fig. 7. The fluorescent properties of 10 were attributed to the chelation of L to the Zn²⁺ centers, which enhanced the rigidity of L. After addition of H₂PO₄⁻ to a CH₃OH solution of sensor 10 remarkable fluorescence quenching was observed with a binding constant of 1.0 × 10⁵ M⁻² (1 : 2 binding mode). This may be a result partly from the electron repelling effect of H₂PO₄⁻ and partly from the decrease of ligand rigidity because of H₂PO₄⁻ binding. Both effects can prohibit the ligand-to-metal charge transfer reducing the fluorescence intensity. Other common anions only caused moderate fluorescence quenching or even enhancement of the fluorescence of sensor 10.

Fig. 7 Proposed binding mode of sensor 10 with H₂PO₄⁻.

3. Intensity-based “turn-on” fluorescent sensors for H₂PO₄⁻

In this section, the advances in “turn-on” H₂PO₄⁻ fluorescent probes will be described. Compared with the “turn-off” type, these are preferable as they circumvent some of the drawbacks of fluorescence quenching, for example, the limited sensitivity and restricted practical applications. Fluorescence enhancement can occur by a number of pathways such as the prohibition of PET (Section 3.1 and Scheme 2b), the increase of rigidity of the sensors (Section 3.2) and “displacement” which releases the fluorescent indicator after addition of H₂PO₄⁻ (Section 3.3). The spectroscopic and analytical parameters of each “turn on” fluorescent sensor for H₂PO₄⁻ have been summarized in Table 2.

Table 2 Spectroscopic and analytical parameters of the “turn on” fluorescent sensors for H₂PO₄⁻

Sensor	Solvent	Fluorophore	λem (nm)	Fluorescent mechanism	H–G stoichiometry	Ka determined from fluorescence	Ref.
11a	CH3CN	Binaphthol	365	PET	1 : 1	1.2 × 10^4 M^−1	38
12	CH3CN	Anthracene	420	PET	1 : 1	—	39
13	CH2Cl2–CH3OH 9 : 1 (v/v)	Anthracene	421	PET	1 : 1	2.8 × 10^3 M^−1	40
14	CH3CN	Anthracene	432	PET, rigidity effect	1 : 2	5.5 × 10^9 M^−2	41
15	DMSO	Schiff-base	333	PET, rigidity effect	1 : 1	—	42
16	CH3CN	Quinoline	393	PET, rigidity effect	1 : 1	3.3 × 10^4 M^−1	43
17	CH3CN	Ru-complex	608	Rigidity effect	1 : 2	7.6 × 10^9 M^−2	44
18	DMSO	Binaphthol	396	Rigidity effect	1 : 1	5.0 × 10^3 M^−1	45
19	CH3CN–MOPS buffer 3 : 1, (v/v)	Naphthalimide	540	Displacement	—	—	46
20	CH3CN	Acridine	422	Additional hydrogen bond	1 : 2	>10^8 M^−2	47
21	DMSO–HEPES buffer 1 : 9 (v/v)	Schiff-base	525	Intramolecular hydrogen bond	1 : 1	2.0 × 10^3 M^−1	48

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3.1 Inhibition of PET

A benzimidazolium-based macrocyclic fluorescent sensor (11a) (Fig. 8) was reported by Ghosh et al.³⁸ Significant fluorescence enhancement of sensor 11a after binding H₂PO₄⁻ was observed in CH₃CN which was assumed to be related to the deactivation of the PET process occurring between the macrocyclic binding domain and the excited state of the BINOL fluorophore. Furthermore, other anions, except fumarate, interacted weakly with sensor 11a. The selectivity and binding affinity of sensor 11a towards H₂PO₄⁻ was greater than the acyclic sensor 11b (K_a = 1.2 × 10⁴ M⁻¹ and 7.5 × 10³ M⁻¹ respectively).

Fig. 8 Structures of sensors 11a and 11b.

By inhibiting the PET process from an anthracene fluorophore to pyridinium moieties, Gong et al.³⁹ developed a H₂PO₄⁻ fluorescent sensor (12) (Fig. 9). The sensor 12 displayed an excellent H₂PO₄⁻ selectivity over the tested common anions in both CHCl₃ and CH₃CN by significant enhancement of monomer emission of anthracene. Interestingly, it was found that during the titration in CHCl₃, two-mode sensing of H₂PO₄⁻ was exhibited: addition of less than equimolar H₂PO₄⁻ induced the enhancement of excimer emission of the anthracene moiety alone, while continuous addition until 3 equiv. of H₂PO₄⁻ made significant enhancement of only monomer emission overwhelming the excimer emission. However, in the polar solvent CH₃CN, only the enhancement of monomer emission was observed during the whole titration. The possible binding process of sensor 12 with H₂PO₄⁻ in CHCl₃ was proposed in Fig. 9.

Fig. 9 Possible binding mode of sensor 12 with H₂PO₄⁻ in CHCl₃.

Recently, Cao et al.⁴⁰ synthesised an anthracene-based fluorescent sensor (13) bearing two 1,2,3-triazolium groups (Fig. 10). The sensor 13 showed efficient fluorescence enhancement after addition of H₂PO₄⁻ in CH₂Cl₂–CH₃OH (9 : 1, v/v) with a binding constant of 2.8 × 10³ M⁻¹, while no obvious change was observed for other common anions. The presence of 100 equiv. of other anions did not cause any significant change for the emission of sensor 13 with 50 equiv. of H₂PO₄⁻. The unique fluorescence enhancement of 13 induced by H₂PO₄⁻ can be attributed to the formation of the (C–H)⁺⋯O interaction which diminished acceptor properties of the triazolium ions inhibiting the PET process from anthracene to the charged triazoliums.

Fig. 10 The structure of sensors 13.

A neutral fluorescent sensor (14) (Fig. 11) based on a calix[4]arene tetraamide derivative and anthracene was synthesised by Chen and co-workers.⁴¹ The fluorescence of sensor 14 was efficiently enhanced about 130% [(I − I₀)/I₀] upon addition of 5 equiv. of H₂PO₄⁻ in CH₃CN. More importantly, it exhibited a high selectivity for H₂PO₄⁻ over a wide range of common anions. The binding constant between 14 and H₂PO₄⁻ was 5.5 × 10⁹ M⁻² with a 1 : 2 stoichiometry. The effective emission enhancement of sensor 14 towards H₂PO₄⁻ is probably due to the inhibition of PET or the increased rigidity.

Fig. 11 Structures of sensors 14–16.

A simple H₂PO₄⁻ fluorescent sensor (15) bearing phenol and thiourea binding sites was developed by Shao and co-workers.⁴² The presence of H₂PO₄⁻ induced the “off-on” fluorescence of 15 in DMSO. This is presumably due to both the inhibition of PET and the binding-induced increased rigidity of the sensor. A distinct colour change was also observed, which could be attributed to the deprotonation of the phenol. The binding constant of 15 with H₂PO₄⁻ was determined to be 5.6 × 10⁴ M⁻¹. Unfortunately, F⁻ and AcO⁻ exhibited significant interference for the detection of H₂PO₄⁻ using sensor 15.

Goswami et al.⁴³ developed a Cu²⁺-based fluorescent sensor (16) for H₂PO₄⁻ in CH₃CN. In the absence of H₂PO₄⁻, the Cu²⁺-complex was in a fluorescence-quenching state. However, when in the presence of H₂PO₄⁻, the emission intensity of the complex increased dramatically, while much smaller increases in intensity were observed for other anions. The binding constant between 16 and H₂PO₄⁻ was measured to be 3.3 × 10⁴ M⁻¹. The metal complex might form a suitable cavity for the selective inclusion of H₂PO₄⁻, as a consequence, the rigidity of the formed complex increased after H₂PO₄⁻ complexation. In addition, the anion binding may also suppress the extent of electron transfer between the quinolines and Cu²⁺ resulting in the fluorescence enhancement.

3.2 Increase of rigidity effect

As mentioned for sensors 14–16, apart from the inhibition of PET, the rigidity effect has been employed to explain the fluorescence enhancement upon anion binding. Indeed the increased rigidity of the formed binding complex could make the non-radiative decay from the excited state less probable and consequently, the emission intensity increases.¹⁷

Ruthenium(II) complex (17) (Fig. 12) has been developed as a selective fluorescent sensor for H₂PO₄⁻ in CH₃CN.⁴⁴ Almost 3-fold fluorescence enhancement of sensor 17 was observed after addition of 10 equiv. of H₂PO₄⁻. The enhanced emission of 17 in the presence of H₂PO₄⁻ was caused by the formation of a hydrogen bond between H₂PO₄⁻ and imidazolyl NH which increased its rigidity and planarity. The binding constant determined by fluorescence was as large as 7.6 × 10⁹ M⁻² (1 : 2 binding mode). In contrast, the same amount of F⁻ and AcO⁻ resulted in significant fluorescence quenching due to the deprotonation of NH, while the changes induced by other common anions were negligible. It is worth mentioning that sensor 17 can also be used as a colorimetric receptor for Fe²⁺ in CH₃CN–HEPES (1 : 71, v/v) and is thus a bifunctional sensor.

Fig. 12 Structures of sensors 17–18.

Huang et al.⁴⁵ designed and synthesised an easily prepared H₂PO₄⁻ fluorescent sensor (18) containing urea group based on binaphthyl. The sensor 18 displayed switch-on fluorescence and the largest binding constant towards H₂PO₄⁻ in DMSO. Upon complexation with H₂PO₄⁻, sensor 18 was rigidified, causing the vibrational and rotational relaxation modes of non-radiative decay inhibited, leading to the increase of emission. Unfortunately, F⁻ and AcO⁻ can also give rise to the fluorescence enhancement with similar binding constants (∼10³ M⁻¹).

3.3 “Displacement”: releasing the fluorescent indicator

A naphthalimide derivative (19) (Fig. 13) was developed as a fluorescent sensor by Chen and co-workers.⁴⁶ The sensor 19 displayed obvious fluorescence quenching in the presence of Cu²⁺ in CH₃CN–MOPS buffer (3 : 1 v/v) due to the inherent paramagnetic nature of Cu²⁺. The formed metal complex exhibited a reversible fluorescence enhancement after addition of H₂PO₄⁻, while the disturbances of other common anions were subtle. The retrievable “off-on” fluorescent behaviour of sensor 19 can be attributed to the release of the fluorescent 19 from the formed Cu²⁺–19 complex after addition of H₂PO₄⁻.

Fig. 13 Sensing mechanism of sensor 19 towards H₂PO₄⁻.

3.4 Other mechanisms

An acridine derivative, bearing two imidazolium groups, as a selective fluorescent sensor (20) (Fig. 14) for H₂PO₄⁻ has been developed by Kim and co-workers.⁴⁷ Significant increase of the fluorescence of 20 was observed after addition of H₂PO₄⁻ in CH₃CN, while fluorescence quenching behaviour was observed for other anions. The binding constant of 20 with H₂PO₄⁻ was measured to be larger than 10⁸ M⁻² with a 1 : 2 stoichiometry. Compared with their previously reported H₂PO₄⁻ “turn-off” fluorescent sensor (1 in Fig. 1), the only difference is the central nitrogen atom on the acridine fluorophore. Thus the authors inferred that the additional hydrogen bond formed between the nitrogen on the acridine moiety and H₂PO₄⁻ afforded the anion-induced fluorescence enhancement.

Fig. 14 Structures of sensors 20 and 21.

Recently, a new fluorescent sensor (21) for H₂PO₄⁻ based on a Schiff-base was reported by Sen and co-workers.⁴⁸ After excitation in the visible region wavelength (480 nm), the sensor 21 could detect H₂PO₄⁻ optically by the unique fluorescence enhancement at 525 nm in DMSO–HEPES buffer (1 : 9, v/v) with a detection limit of 3.5 × 10⁻⁶ M. The binding constant was determined to be 2.0 × 10³ M⁻¹. The response was due to the formation of the intermolecular hydrogen bonds between H₂PO₄⁻ and sensor breaking the intramolecular hydrogen bonding network between the three phenol residues. In addition, the presence of other common anions did not affect the detection of H₂PO₄⁻. Bio-imaging studies indicated that sensor 21 is an efficient staining agent and could be used for monitoring intracellular H₂PO₄⁻.

4. Ratiometric fluorescent sensors for H₂PO₄⁻

Realizing ratiometric fluorescent sensing of H₂PO₄⁻, which involves the ratio of fluorescence intensities at two different wavelengths before and after H₂PO₄⁻ binding, is a current research focus. Compared with the “turn-off” or “turn-on” fluorescent sensors, ratiometric fluorescent probes present several advantages, for example they permit signal rationing and thus increase the dynamic range and provide built-in correction for environmental effects. In addition, the distinct fluorescent colour change after anion complexation will be practically useful for both visual sensing and convenient bio-imaging of anions. A typical mechanism affording this phenomenon is the formation or extinction of an intramolecular (Section 4.1, Scheme 3a and Table 3) or intermolecular (Section 4.2, Scheme 3b and Table 4) excimer. This will give rise to a change of the intensity ratio between monomer and excimer emissions of the fluorophore based upon the amount of the added anions. ESPT and other principles will also be described in Section 4.3 and 4.4 (Table 5).

Scheme 3 Diagrams for (a) the intramolecular excimer formation and (b) the intermolecular excimer formation after anion binding.

Table 3 Spectroscopic and analytical parameters of the ratiometric H₂PO₄⁻ fluorescent sensors following the mechanism of intramolecular excimer

Sensor	Solvent	Fluorophore	λmonomer emission (nm)	λexcimer emission (nm)	H – G stoichiometry	Ka determined from fluorescence	Ref.
22	CH3CN	Naphthalene	350	456	1 : 1	9.1 × 10^3 M^−1	49
23	CH3CN	Anthracene	419	525	1 : 1, 1 : 2	5.4 × 10^3 M^−1	50
24	CH3CN–DMSO 99–1 (v/v)	Anthracene	418	500	1 : 1	3.1 × 10^4 M^−1	51
25	CHCl3–CH3CN 98 : 2 (v/v)	Anthracene	413	513	1 : 1, 1 : 2	1.2 × 10^4 M^−1	52
26	CH3CN	Pyrene	403	482	1 : 1	2.2 × 10^4 M^−1	53
27a	CH3CN	Anthracene	412	507	1 : 2	2.5 × 10^4 M^−1, 2.2 × 10^4 M^−1	54
28	CH3CN	Anthracene	420	485	1 : 1	2.2 × 10^5 M^−1	55
29	CH3CN	Anthracene	418	500	1 : 1, 1 : 3	—	57
30	THF	Pyrene	377	477	1 : 1	1.9 × 10^5 M^−1	58
31	THF	Pyrene	375	477	1 : 2	—	59
32	CH3CN	Pyrene	380	483	1 : 1	—	60
33	CH3CN–CH2Cl2–H2O 1000 : 1 : 5 (v/v/v)	Pyrene	396	485	1 : 1	1.0 × 10^5 M^−1	61

---

Table 4 Spectroscopic and analytical parameters of the ratiometric H₂PO₄⁻ fluorescent sensors following the mechanism of intermolecular excimer

Sensor	Solvent	Fluorophore	λmonomer emission (nm)	λexcimer emission (nm)	H – G stoichiometry	Ka determined from fluorescence	Ref.
34	CHCl3–DMSO 99.9 : 0.1 (v/v)	Naphthalene	338	420	1 : 1	—	63
35	HEPES buffer	Anthracene	432	490	1 : 1	>4.6 × 10^5 M^−1	64
36	CH3CN	Anthracene	429	500	1 : 1	3.0 × 10^6 M^−1	65
37	CH3CN	Pyrene	400	493	1 : 1	2.4 × 10^6 M^−1	66
38a	CH3CN	Acridine	430	480	1 : 1	5.1 × 10^4 M^−1	67
39	CH3CN	Acridine	430	556	1 : 1	—	68
40	CH3CN	Acridine	430	544	1 : 1	—	68
41	CH3CN	Acridine	430	503	1 : 1	—	68
42	CH3CN	Acridine	430	466	1 : 1	—	68
43	CH3CN	Acridine	430	501	1 : 1	2.9 × 10^6 M^−1	69
44a	CH3CN	Anthracene	428	500	1 : 1	2.6 × 10^5 M^−1	69

---

Table 5 Spectroscopic and analytical parameters of the ratiometric H₂PO₄⁻ fluorescent sensors following other mechanisms

Sensor	Solvent	Fluorophore	Original λem (nm)	Induced λem (nm)	H–G stoichiometry	Ka determined from fluorescence	Ref.
45	DMSO–1,4-dioxane 1 : 1 (v/v)	Coumarin	430	540	1 : 1	2.0 × 10^6 M^−1	70
46	CH3CN–H2O 9 : 1 (v/v)	Naphthalene	343	412	1 : 1	1.0 × 10^5 M^−1	74
47	CHCl3	Acridine	420	510	—	—	75
48	CH3CN–DMSO 99.99 : 0.01 (v/v)	Azaindole	365	480	1 : 1	2.0 × 10^4 M^−1	76
49	CH3CN–DMSO 99.99 : 0.01 (v/v)	Azaindole	365	480	1 : 1	1.1 × 10^4 M^−1	77
50	CH3CH2OH–THF 3 : 1 (v/v)	Hexaphenylbenzene	438	366	—	—	78

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4.1 Intramolecular excimer formation/extinction

A series of tweezer-like fluorescent sensors (22–27) (Fig. 15 and 16) have been developed by Ghosh and co-workers.^49–54 These sensors utilized an intramolecular excimer based on either naphthalene, anthracene or pyrene fluorophores. By incorporating benzimidazolium or amide moieties as anion binding sites, these sensors could achieve ratiometric sensing of H₂PO₄⁻ either in organic or aqueous media with moderate binding constants (∼10⁴ M⁻¹) but high sensing selectivity.

Fig. 15 Structures of sensors 22–26.

Fig. 16 Structures of sensors 27a and 27b.

The ortho-phenylenediamine based sensor 22 (ref. 49) could selectively bind H₂PO₄⁻ in CH₃CN by exhibiting excimer emission at 456 nm due to the π–π stacking between two pendant naphthalene fluorophores. The sensor 23,⁵⁰ based on the same ortho-phenylenediamine unit, could recognize H₂PO₄⁻ in CH₃CN displaying a significant decrease of monomer emission of anthracene, along with a weak increase of excimer emission at 525 nm. A ratiometric change in emission after addition of H₂PO₄⁻ was also achieved by the enediyne scaffold based sensor 24 (ref. 51) in CH₃CN containing 1% DMSO. Compared with 23, in spite of the absence of the amide moieties as hydrogen bonding sites in sensor 24, the binding constant of sensor 24 with H₂PO₄⁻ was larger than that of 23 (5.4 × 10³ vs. 3.1 × 10⁴ M⁻¹) possibly because the linear nature of the triple bonds in 24 make the sensor more rigid. Pyridinium amide-based sensor 25 (ref. 52) built on a biphenyl scaffold showed selective complexation of H₂PO₄⁻ with the enhancement of both monomer and excimer emission at 413 and 513 nm respectively in CHCl₃ containing 2% CH₃CN. By utilizing pyrene as the signalling unit, the benzimidazolium-based sensor 26 (ref. 53) was synthesised and used as an efficiently ratiometric fluorescent sensor for H₂PO₄⁻ in CH₃CN due to its capacity of forming an excimer with pyrene.

An anthracene-labeled 1,2,3-triazole-linked bispyridinium sensor (27a)⁵⁴ (Fig. 16) was also developed as a ratiometric fluorescent sensor for H₂PO₄⁻ over other common anions in CH₃CN. Job's plot analysis demonstrated a 1 : 2 stoichiometry between host and guest, and the binding constants were 2.5 × 10⁴ M⁻¹ and 2.2 × 10⁴ M⁻¹ for the successive complexation of the guest molecules. Additionally, the sensor 27a could form a stable gel after addition of H₂PO₄⁻ in CHCl₃–CH₃CN (9 : 1, v/v), which also provided a convenient approach for visually sensing H₂PO₄⁻. Interestingly, the control sensor 27b without the triazole links failed to form the gel with H₂PO₄⁻, highlighting the crucial role played by the triazole links on gel formation and H₂PO₄⁻ recognition.

Xu et al.⁵⁵ also developed a structurally similar ratiometric fluorescent sensor (28) (Fig. 17) for H₂PO₄⁻ in CH₃CN based on imidazolium and anthracene groups. The sensor 28 displayed a unique excimer peak at 485 nm only in the presence of H₂PO₄⁻ with a binding constant of 2.2 × 10⁵ M⁻¹. The addition of 10 equiv. of other anions did not cause any obvious change to the emission of 28 with H₂PO₄⁻ (2 equiv.). The rigid phenanthroline moiety in sensor 28 allowed specific hydrogen bonds with H₂PO₄⁻ accounting for the remarkable selectivity observed.

Fig. 17 The structure of sensor 28.

Most H₂PO₄⁻ ratiometric fluorescent sensors involving the formation of an intramolecular excimer contain two appended rigid fluorophores. However some sensors bearing multiple fluorophore substituents have been reported. It was believed that tripodal shaped receptors with anion binding groups attached on the three pendant arms would be able to complex anions effectively, due to their preferable pre-organization and conformational flexibility.⁵⁶ Thus Ghosh et al.⁵⁷ synthesised a new benzimidazolium-based tripodal fluorescent sensor (29) (Fig. 18) bearing three pendant anthracene rings. The sensor 29 exhibited a high selectivity towards H₂PO₄⁻ among all the common anions in CH₃CN via significant quenching of monomer emission (418 nm) along with the formation of a weak excimer at 500 nm.

Fig. 18 Structures of sensors 29–31.

A two-arm pyridine amide sensor (30) (Fig. 18) bearing four pyrenes as signal units was synthesised by Liao and co-workers.⁵⁸ This sensor, which provided a pseudo-tetrahedral cleft and multiple hydrogen bonds, formed a 1 : 1 complex with H₂PO₄⁻ in THF leading to a significant decrease of the emission intensity ratio between the pyrene monomer (377 nm) and the excimer (477 nm). Further experiments demonstrated that the excimer emission of 30 was formed between two pyrene rings in different arms. Subsequently, a similar two-arm compound (31) based on ferrocene was developed as a ratiometric fluorescent sensor for H₂PO₄⁻ by the same group.⁵⁹ The introduction of ferrocene offered another approach, i.e. cyclic voltammetry, for investigation of anion binding. Interestingly, in this case, the two-arm ferrocene hexamide sensor 31 formed a complex with H₂PO₄⁻ with 1 : 2 stoichiometry: with each arm bound to one H₂PO₄⁻ molecule independently. A synclinal conformation was adopted by sensor 31 in THF which was further stabilized by complexation with H₂PO₄⁻.

The above-documented H₂PO₄⁻ ratiometric sensors mostly relied on hydrogen bonding interactions. However, taking advantage of the ion-pairing electrostatic interaction between a metal ion and an anion, would also be useful as this would permit simultaneous binding of cationic and anionic species. It would also allow for a potential co-operative effect to be investigated, in which the anion/cation binding ability of the sensor could be significantly enhanced when in the presence of a bound cation/anion.

A fluorescent sensor (32) (Fig. 19a) bearing pyreneamides as anion binding sites and a signalling unit based on calix[4]crown-5 has been designed and synthesised by Choi and co-workers.⁶⁰ The fluorescent excimer emission of 32 was first significantly increased after addition of K⁺ in CH₃CN. Subsequently, it was observed that the excimer emission between the two pyrenes of 32 K⁺ declined obviously upon subsequent addition of H₂PO₄⁻, while no change for other common anions occurred. The fact that the 32 K⁺ complex could achieve ratiometric sensing of H₂PO₄⁻ while 32 cannot demonstrated that the binding of K⁺ for 32 favoured the following association with H₂PO₄⁻ as illustrated in Fig. 19a, i.e. the ion-pairing electrostatic interaction between K⁺ and H₂PO₄⁻ played an important role in the sensing behaviour. However, it cannot be neglected that to saturate sensor 32 a large excess of H₂PO₄⁻ was required (10 000 fold) and the K⁺ coordinated complex might be decomposed at such high concentrations of H₂PO₄⁻.

Fig. 19 Structures of sensors 32 and 33, and their binding modes with metal ions and H₂PO₄⁻.

A Similar fluorescent sensor 33 (Fig. 19b) based on a pyrene-linked triazole-modified homooxacalix[3]arene was synthesised by Ni and co-workers.⁶¹ The sensor showed ratiometric sensing of Zn²⁺ by enhancing the monomer emission while weakening the excimer in CH₃CN–CH₂Cl₂–H₂O (1000 : 1 : 5, v/v/v). Interestingly, the subsequent addition of H₂PO₄⁻ to the solution of 33 Zn²⁺ resulted in a reversed ratiometric fluorescent change with a detection limit of 1.52 × 10⁻⁷ M. The binding constant was determined to be 1.0 × 10⁵ M⁻¹. The observation that only small fluorescence intensity changes of monomer and excimer emissions of 33 were induced by H₂PO₄⁻ indicated that Zn²⁺ played a key role in sensing of H₂PO₄⁻. Furthermore, there is no interference for the detection of 40 equiv. of H₂PO₄⁻ when in the presence of 40 equiv. of other anions.

4.2 Intermolecular excimer formation/extinction

Apart from intramolecular π–π stacking between two appended fluorophores, the intermolecular excimer which occurs between molecules is also a widely used strategy for designing ratiometric sensors. The change of the intensity ratio between the monomer and excimer emissions of the sensor when in the presence of a certain equivalent of anions in various concentrations can confirm whether the formation of excimer is intramolecular or intermolecular. If the values remain constant at different concentrations, the anion-induced excimer occurs intramolecularly. However if there is a change in the ratio, the excimer is being formed intermolecularly.⁶²

A naphthalene-based 2,5-diketopiperazine (34) (Fig. 20) was developed as a ratiometric fluorescent sensor for H₂PO₄⁻ in CHCl₃ containing 0.1% DMSO.⁶³ The monomer emission (338 nm) of sensor 34 was dramatically quenched when in the presence of H₂PO₄⁻ along with the appearance of a weak excimer peak at 420 nm. The binding constant was measured to be 4.7 × 10² M⁻¹ with UV method. The H₂PO₄⁻-induced excimer was assigned to be an intermolecular interaction, since the bifunctional hydrogen bonding donor sites in H₂PO₄⁻ are able to bring two naphthalene residues into close proximity by forming hydrogen bonds with the oxygen atoms between two neighboring diketopiperazines. Unfortunately, aliphatic dicarboxylic acids, such as malonic, succinic, glutaric and adipic acids, could also give rise to the similar fluorescent behaviour, showing the poor selectivity of the sensor.

Fig. 20 Structures of sensors 34–36.

As metal–ligand receptors have a strong binding affinity for anions in competitive media, Huang et al.⁶⁴ synthesised an anthracene derivative (35) containing Zn²⁺ sites as a ratiometric fluorescent sensor for H₂PO₄⁻. This sensor showed a selective fluorescence enhancement of the excimer emission at 490 nm as well as decrease of monomer emission at 432 nm with H₂PO₄⁻ in 0.01 M HEPES buffer, pH = 7.4. The binding constant was measured to be larger than 4.6 × 10⁵ M⁻¹ by fluorescence. The formation of the anthracene dimer between two molecules induced by H₂PO₄⁻ was attributed to binding of H₂PO₄⁻ within the four zinc binding sites as well as due to a favorable π–π interaction between two flat hydrophobic anthracene rings. Other common anions except SO₄²⁻ only cause a minor fluctuation in monomer emission. In the case of SO₄²⁻, a weak excimer emission could be induced when 10 equiv. were added.

A similar anthracene derivative (36) bearing amidopyridinium groups as anion binding sites was developed as a fluorescent sensor for H₂PO₄⁻.⁶⁵ A concentration-dependent experiment showed that a new excimer peak was observed at high concentration of 36, demonstrating that the sensor has a tendency to aggregate in solution. The anion sensing results revealed that among all the tested anions, only H₂PO₄⁻ could induce the strong excimer emission of 36 in CH₃CN with a binding constant of 3.0 × 10⁶ M⁻¹, which can be attributed to H₂PO₄⁻-templated assembly of sensor molecules forming the anthracene excimer. The detection limit was determined to be 3.62 × 10⁻⁷ M.

Taking advantage of the strategy of anion-induced intermolecular π–π stacking, recently, in our group we also synthesised a series of acyclic and macrocyclic H₂PO₄⁻ ratiometric fluorescent sensors bearing benzimidazolium and urea groups as binding sites built on pyrene, acridine or anthracene fluorophore.^66–69

Initially, the synthesis of a flexible pyrene-based fluorescent sensor 37 (Fig. 21) bearing benzimidazolium and urea groups was achieved.⁶⁶ This sensor was able to distinguish H₂PO₄⁻ from other anions in CH₃CN by displaying ratiometric fluorescent sensing behaviour. Even though there are two pyrene rings per sensor molecule, the pyrene excimer was formed through an intermolecular pattern as illustrated in Fig. 21. This was demonstrated by the change of the intensity ratio of I_E/I_M which varied with the concentration of sensor 37 when in the presence of 2 equiv. of H₂PO₄⁻. The detection limit of H₂PO₄⁻ was achieved as 5.02 × 10⁻⁷ M. A synergistic effect between benzimidazolium and urea moieties can account for the high binding constant obtained (K_a = 2.4 × 10⁶ M⁻¹).

Fig. 21 The binding mode of sensor 37 with H₂PO₄⁻.

To investigate the synergistic binding effect more accurately, we further designed an acridine derivative (38a) containing benzimidazolium and urea moieties (Fig. 22).⁶⁷ For comparison, sensors 38b and 38c which only possess one type of binding site were prepared. Sensing results showed that 38a could be used as a ratiometric fluorescent sensor for H₂PO₄⁻ in CH₃CN by exhibiting a significant decrease of monomer emission at 430 nm and increase of the excimer at 480 nm with a binding constant of 5.1 × 10⁴ M⁻¹. In contrast, the ratiometric sensing behaviours of sensors 38b and 38c toward H₂PO₄⁻ were rather poor. A 2 : 2 binding complex was proposed between 38a and H₂PO₄⁻, which was directly detected by HRMS analysis. In addition, the sensor 38a was also able to detect HSO₄⁻ in CH₃CN according to obvious fluorescence quenching (K_a = 1.7 × 10⁶ M⁻¹), while almost no response towards other anions was observed.

Fig. 22 Structures of sensors 38a–c.

Macrocyclic anion receptors tend to display higher binding constants than their acyclic counterparts due to their well-preorganized topology. Therefore, we synthesised a series of acridine derived benzimidazolium macrocyclic sensors (39–42) (Fig. 23).⁶⁸ X-ray crystal structures revealed that these sensors had a tendency to aggregate forming acridine dimers. Anion binding studies showed that all the four sensors displayed “turn-on” as well as a bathochromic-shift in fluorescence emission only in the presence of H₂PO₄⁻ in CH₃CN. Furthermore, different cavity size (39, 40 vs. 41) or rigidity (40 vs. 42) of the sensors exhibited different bathochromic-shifts (from 36 to 126 nm) giving rise to various fluorescent colour changes. The tunable bathochromic-shifted emissions of these sensors induced by H₂PO₄⁻ were attributed to anion-directed assembly of sensors forming the acridine excimers to a different extent.

Fig. 23 Structures of sensors 39–42 and their binding modes with H₂PO₄⁻.

In order to further improve the binding affinities of the macrocyclic sensors 39–42 with H₂PO₄⁻, a urea moiety was then introduced. The aim being that the urea binding site would synergistically bind H₂PO₄⁻ along with benzimidazolium moiety.⁶⁹ Results showed that compared with sensors 39–42, the sensor 43 (Fig. 24) had a better ratiometric sensing performance toward H₂PO₄⁻ in CH₃CN, such as a higher binding constant (2.9 × 10⁶ M⁻¹) and lower detection limit (1.0 × 10⁻⁶ M). In addition, no significant variation in the intensity ratio (I₅₀₁/I₄₃₀) was found for the detection of 2 equiv. of H₂PO₄⁻ when in the presence of 20 equiv. of other anions. Furthermore, interesting results were also achieved from a structurally similar anthracene cyclophane (44a) which exhibited an improved anion binding performance toward H₂PO₄⁻ compared to the sensor 44b, which incorporated only benzimidazolium groups (K_a = 2.6 × 10⁵ M⁻¹ and 1.6 × 10⁵ M⁻¹ respectively).

Fig. 24 Structures of sensors 43, 44a and 44b.

4.3 Intermolecular excited-state proton transfer

Excited-state proton transfer (ESPT) is an efficient approach utilized in the design of the ratiometric fluorescent sensors. The ESPT process generally incorporates a fast excited-state proton transfer from a proton donor (usually hydroxyl or amino group) to an acceptor group (often either oxygen or nitrogen) mediated by a hydrogen bond. In addition, a large apparent Stokes shift can be observed, which makes it very suitable for designing ratiometric fluorescent sensors. A specific illustration involving both PCT and ESPT can be seen in Scheme 4, apart from the emission channel from CT excited state, the proton transfer from the fluorophore to the anion can open the second emission channel, thus resulting in a ratiometric sensing behaviour.⁷⁰

Scheme 4 The process of intermolecular excited state proton transfer between the receptor and anion.

With the strategy shown in Scheme 4, in 2001, Choi et al. reported the first ESPT-based ratiometric fluorescent sensor (45) (Fig. 25) built on the macrocyclic amide containing a coumarin fluorophore.⁷⁰ In DMSO–1,4-dioxane (1 : 1, v/v), the sensor 45 showed preferential binding towards tetrahedral anions, especially for H₂PO₄⁻ with a binding constant of 2.0 × 10⁶ M⁻¹. In addition to a weak increase of the initial CT band at 430 nm after addition of H₂PO₄⁻, a remarkable new emission band at 540 nm was observed. This was argued to be induced by proton transfer from the coumarin excited state to H₂PO₄⁻. The intensity of the second emission band was related with the basicity of the anions tested. It should be noted that after this case, further examples using this mechanism have been reported for the detection of basic anions such as F⁻ and AcO⁻.^71–73

Fig. 25 The structure of sensor 45.

4.4 Other mechanisms

The receptor 46 (Fig. 26) bearing theophyllinium as the key binding motif and a naphthalene moiety as the sensing unit was synthesised by Mahapatra and co-workers.⁷⁴ This sensor could selectively recognize H₂PO₄⁻ in CH₃CN–H₂O (9 : 1, v/v) by exhibiting a significant decrease of emission at 343 nm and increase of a broad emission at 412 nm with a binding constant of 1.0 × 10⁵ M⁻¹. The appearance of the new fluorescence band at longer wavelength (412 nm) might be attributed to the ‘conformational restriction’ after anion binding or the inhibition of PET via charge transfer between the excited state of naphthalene and theophyllium binding sites. For other common anions, the emission of 46 was decreased to a small extent.

Fig. 26 Structures of sensors 46–49.

Martí-Centelles et al.⁷⁵ synthesised a macrocyclic fluorescent sensor (47) based on acridine fluorophore. Upon addition of various acids in CHCl₃, there is an increase in emission at 510 nm while the original emission at 420 nm disappeared only in the case of H₃PO₄. The fluorescent recognition of H₃PO₄ via a bathochromic-shift was attributed to the supramolecular interactions between H₂PO₄⁻ and the strongly fluorescent acridinium ion generated by the acid protonation of 47. This assumption was supported by the fact that no change in fluorescence was observed when 47 was titrated with H₂PO₄⁻ or TFA alone, but the fluorescence significantly increased when simultaneously adding both TFA and H₂PO₄⁻.

Recently, Ghosh et al. developed two azaindole-1,2,3-triazole based ratiometric fluorescent sensors 48 (ref. 76) and 49 (ref. 77) for H₂PO₄⁻ in CH₃CN containing 0.01% DMSO. Both of the two sensors selectively exhibited a decrease in emission at 365 nm and a weak increase at 480 nm upon H₂PO₄⁻ binding. The binding constants of 48 and 49 with H₂PO₄⁻ were determined to be 2.0 × 10⁴ M⁻¹ and 1.1 × 10⁴ M⁻¹ respectively. The fluorescence quenching at 365 nm was argued to be due to a reduction in the difference between the lowest energy singlet excited state (nπ*) and the ground singlet state after H₂PO₄⁻ complexation. The appearance of the peak at 480 nm was attributed to the increase in conjugation between azaindoles and triazoles. In addition, the sensor 49 could be used as an efficient “turn-on” fluorescent sensor for Cl⁻ in the same solvent. Furthermore, for practical applications, the sensor 48 showed no selectivity towards ATP, ADP and AMP in CH₃CN containing 0.01% DMSO–H₂O (4 : 1, v/v), while 49 could selectively recognize ATP over ADP and AMP and was used for the bio-imaging of ATP in Hela cells.

Bhalla et al.⁷⁸ designed and synthesised a hexaphenylbenzene-based derivative (50) (Fig. 27). This sensor showed fluorescence enhancement at 438 nm in the presence of Zn²⁺ in ethanol–THF (3 : 1, v/v) due to the suppression of PET from the imino nitrogens to the hexaphenylbenzene scaffold. Additionally, the Zn²⁺ ensemble of compound 50 exhibited a selective fluorescent response towards H₂PO₄⁻: the emission band at 438 nm was quenched and a new hypochromatic-shifted band at 366 nm appeared. The fluorescent phenomenon was attributed to the weakening of the existing 50–Zn²⁺ bonds due to the interaction of H₂PO₄⁻ with Zn²⁺. The detection limit was 10⁻⁸ M. In the case of other anions, no significant change in emission was observed except with AMP which induced the enhancement of emission along with a slight hypochromatic-shift from 438 to 431 nm.

Fig. 27 The structure of sensor 50.

5. Concluding remarks

In this review, H₂PO₄⁻ fluorescent sensors based on synthetic organic molecules have been discussed. These sensors fluorescently detect H₂PO₄⁻ utilizing either “turn-off”, “turn-on” or ratiometric sensing behaviour. Promotion/inhibition of PET, binding-induced rigidity effect and intra/inter-molecular excimer formation/extinction are the most commonly employed principles involved in the process of H₂PO₄⁻ recognition. Ratiometric fluorescent sensors utilizing the intra/inter-molecular excimer formed between fluorophores have been developed as the most successful strategy and are a current focus for future H₂PO₄⁻ sensing.

Careful design of the binding site and structure has allowed the development of sensors that can selectively distinguish H₂PO₄⁻. This can be achieved even in the presence of basic anions, such as F⁻ and AcO⁻, and structurally similar tetrahedral anions such as HSO₄⁻. However an even greater challenge, which still remains, is to selectively sense H₂PO₄⁻ among inorganic/bio-phosphates such as P₂O₇⁴⁻, ATP and ADP. In addition, most sensors utilize hydrogen bonding and so are only effective in organic solvents. Even though there are some metal complex based sensors that realize the recognition in water, the development of H₂PO₄⁻ selective sensors for detection in aqueous media, is a major challenge for chemists. Furthermore, almost all of the reported sensors are only able to achieve qualitative detection of H₂PO₄⁻ rather than quantitative determination. In spite of these deficiencies, science is advancing step by step, and so it can be expected that in the near future a relatively simple optical method for the selective, quantitative detection of the biologically important anion H₂PO₄⁻ in aqueous solution will be available. This will be an important advance, as it may lead to a greater understanding of processes occurring within living cells and organs.

Acknowledgements

The authors are grateful to financial support from the National Natural Science Foundation of China (Grant no. 21072061) and Shanghai Leading Academic Discipline Project (Grant no. B409).

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