An overview of the recent developments on Hg²⁺ recognition

Abstract

Adverse influences of mercury on living organisms are well known. Despite efforts from various regulatory agencies, the build-up of Hg²⁺ concentration in the environment is of serious concern. This necessitates the search for new and efficient reagents for recognition and detection of Hg²⁺ in environmental samples as well as for application in diagnostics. Among various detection processes adopted for designing such reagents, generally methodologies that allow associated changes in spectra properties are preferred for the obvious ease in the detection process. Significant changes in the electronic spectral pattern in the visible region of the spectrum also induce detectable changes in solution colour for naked-eye detection and are useful for developing reagents for in-field sample analysis with yes–no type binary responses. However, reagents that allow detection of Hg²⁺ with associated fluorescence on response are useful for detection of Hg²⁺ in environmental samples, as well as for use as an imaging reagent, for detection of cellular uptake. High spin–orbit coupling constant for Hg²⁺ along with its high solvation energy in aqueous medium poses a challenge in developing efficient reagents with fluorescence on response that work in aqueous medium/physiological condition. To get around this problem, several methodologies, like conversion of rhodamine derivative spirolactam to strongly fluorescent xanthenes that form on binding to Hg²⁺, chemodosimetric reaction for generation of a new luminescent derivative, have been adopted. Apart from these, modified charge transfer processes on binding to Hg²⁺ have also been utilized for designing reagents for optical detection of Hg²⁺. Immobilization of such reagents on solid surfaces also led to the development of self-indicating Hg²⁺ ion scavengers. All such examples are discussed in the present review.

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Introduction

Mercury in its various forms is known to be the most potent neurotoxin for human physiology and its detrimental influences on human life, other living organisms and the surrounding environment are amply described in various contemporary studies. Despite efforts of different regulatory agencies, as well as governmental policies in various countries, for curbing the mercury emission in the environment, the global mercury contamination from natural and industrial processes has posed a serious threat to the human race in the last few decades.¹ The concern over its deleterious effects on various living organisms and the surrounding environment has actually led chemists and more broadly the sensing community to develop new mercury detection methods that are cost-effective, rapid, facile and applicable to the environmental and biological milieus.² All these have triggered an exponential surge in interest for developing an efficient receptor for recognition and detection of Hg²⁺ in aqueous medium and in physiological conditions. This has consequently resulted in innumerable publications from various research groups globally in last one decade. There are review articles on the general theme of metal-ion recognition and sensing, and a few among these have also discussed about possible application potential of such receptors for treating environmental and biological samples as well as use as imaging reagents.³ A closer scrutiny of all these review articles reveals that there has been only one review article by Lippard et al. that has discussed specifically about the various approaches for optical detection of Hg²⁺ in environmental and biological samples.¹ Apart from this, there has been no specific review article that has covered recent advances achieved in developing reagents that are efficient for specific recognition of Hg²⁺ under physiological condition, as well as in using such reagents, as biomarkers or imaging reagents for in vivo studies. This review article aims to discuss such articles with specific emphasis on receptors that exhibit ‘yes’ on specific binding or reaction of Hg²⁺ ion and use of such reagents for analysis of environmental samples as well as use as biomarker/imaging reagent. Design of such receptors depends primarily on the nature of the binding site for achieving the desired specificity for the Hg²⁺ ion, as well as on the nature of the optical response(s), which is/are preferred for probing the receptor–analyte binding process. Among various photophysical pathways, like charge transfer (CT),⁴ electron transfer (ET),⁵ energy transfer (eT),⁶ Förster resonance energy transfer (FRET),⁷ through bond energy transfer (TBET),⁸ are generally utilized for reporting the binding-induced phenomena, while photoactive moieties, like 1,8-napthalimide, coumarin, pyrene, anthracene, BODIPY, squaraine, xanthanes, cyanine, rhodamine, fluorescein, are commonly being used as the reporter moiety.⁹ Apart from these, optical responses, more specifically the luminescence responses associated with receptor–Hg²⁺ binding, on account of change(s) in conformational flexibility, heavy atom effect and dye displacement methodologies are also discussed in this present article.¹⁰

Bio-activity of Hg²⁺ ions

For centuries, mercury was used as an important component for various medicines such as diuretics, antibacterial agents, antiseptics, and laxatives. However, adverse role of mercury on human physiology on prolonged exposure was realized over the years and now mercury is being considered as one of the most potent neurotoxins for humans and mammals. Perhaps, the most deadly form of mercury is organic mercury compounds compared to two other forms of mercury, namely, elemental mercury and inorganic salts, which are generally available in the environment. Organic mercury compounds, more specifically methylmercury, are accumulated in the food chain. Industrial effluents often contain mercury in the inorganic form; however, anaerobic methylation of inorganic mercury by microorganisms and abiotic chemical processes in sediments generates methylmercury. Reports also suggest that vegetation in water bodies also convert inorganic mercury to methylmercury. In water bodies, methylmercury bioaccumulates in living aquatic organisms and biomagnifies up the food chain (Fig. 1). Methylmercury is better absorbed and shows a higher mobility in the human body. It easily passes through biological membranes, such as skin, respiratory, and gastrointestinal tissues, which eventually damages the central nervous and endocrine systems.¹¹

Fig. 1 Scheme showing the bio-accumulation and magnification of CH₃–Hg²⁺ along the food chain.

Considering its acute toxicity, international regulatory agencies, like, environmental protection agency (EPA) in USA have set an upper limit of 2 ppb (10 nM) for Hg(II) in safe drinking water.¹ Due to the well-known toxicity of the Hg²⁺ towards living organisms and the very stringent norms set more recently by the various regulatory agencies, it has become almost imperative to develop suitable colorimetric- or fluorescence-based sensors for water quality determination in terms of the Hg²⁺ ion concentration, and this has actually led to a surge of interest among researchers for designing Hg²⁺ ion-specific sensor that works in aqueous medium, as well as under the physiological condition, which allows the detection of Hg²⁺ ion uptake in lower organisms.

Colorimetric sensors for Hg²⁺ ion

Design of the colorimetric chemosensor for Hg(II) ion has gained interest as this allows simple ‘in-field’ visual detection and semi-quantitative analysis. This simplicity in the detection process is the primary reason for the present emphasis on the development of the colorimetric sensors for Hg²⁺ ion.

Li et al. reported a heptamethine cyanine dye containing dithia-dioxa-monoaza crown ether moiety (1) for the detection of Hg²⁺.¹² Spectral change for this reagent on specific binding to Hg²⁺ in methanol medium appeared in the NIR region of the spectrum (Fig. 2). Spectral changes in the NIR region have a special significance; as biological background is supposed to induce less interference when detecting in the red to NIR (700–1000 nm) region compared with detection in the UV and visible region. A large (∼122 nm) red shift occurs in the absorption spectrum (λ_max shifted from 695 nm for 1 to 817 nm for Hg²⁺·1) with a visually detectable change in colour from blue to colourless. A moderate binding constant (K_a = 4.335 × 10⁴ M⁻¹) was evaluated for 1 : 1 binding stoichiometry based on the electronic spectral titrations.

Fig. 2 (A) Molecular structure of the chemosensor 1 and (B) naked-eye colour change of the solution of 1 in presence of different metal ions. Reprinted with permission from ref. 12. Copyright 2008 American Chemical Society.

Lee and co-workers reported two azo-coupled macrocyclic ionophores having benzene (2) or pyridine (3) subunits (Fig. 3).¹³ Both receptors showed selectivity towards Hg²⁺ and Job’s plot analysis confirmed a 1 : 1 (2/3 : Hg²⁺) stoichiometry for complexation. However, more significant cation-induced hypsochromic shift was observed for 2, suggesting that the presence of the pyridine unit in 3 could have inhibited the Hg²⁺⋯N-azo interaction.

Fig. 3 Molecular structure of chemosensors (A) 2, 3 and (B) 4; (C) change in solution colour of 4 on binding to Hg(II) in 1 : 10 EtOH–H₂O (pH 7, HEPES buffer). Reprinted with permission from ref. 14. Copyright 2006 American Chemical Society.

A hemicyanine dye 4 (Fig. 3), consisting of an aniline donor and benzothiazolium acceptor, was reported by Palomares and co-workers for colorimetric detection of Hg(II) in mixed EtOH–H₂O (1 : 10, v/v) at neutral pH.¹⁴ On selective binding to Hg²⁺, 4 produced a colour change from pink to green (Fig. 3) on formation of a 1 : 1 complex with Hg²⁺ with an associated blue-shift in absorption maxima from ∼550 to ∼450 nm (isosbestic point at ∼480 nm). Interestingly, the evaluated binding constant was ∼10⁷ M⁻¹ even in mixed aqueous medium.

Upadhyay and co-workers reported a ninhydrin-based colorimetric molecular switch (5) (Fig. 4), which gets ‘ON’ (blue) in the simultaneous presence of Hg²⁺ and CH₃COO⁻/F⁻ while the absence of any one of these leads the system ‘OFF’ (purple) in ethanol–water (1 : 1, v/v) medium.¹⁵ The corresponding spectral responses (shift from 535 to 590 nm in the CT band of receptor 5) could be correlated for demonstrating the AND logic function.

Fig. 4 Structures of the chemosensors 5–9.

Tew et al. reported a series of terpyridine derivatives (6–9) (Fig. 4), which selectively detect Hg²⁺ in DMSO–water (1 : 3.5, v/v) medium.¹⁶ These compounds turned into pink colour with Hg²⁺, whereas a pale blue colour was observed with Cu²⁺. For reagent 6, the lowest detection limit for Hg²⁺ was 2 ppm for naked eye, while it was 2 ppb (USEPA limit for Hg²⁺ in drinking water) through spectrophotometry. Crystallographic and isothermal titration calorimetry (ITC) studies proved the 2 : 1 binding stoichiometry for [6]·Hg²⁺ formation. Furthermore, the paper strip developed using 9 could detect Hg²⁺ over a wide pH range (2.5 to 9) demonstrated its practical applicability.

Sun et al. synthesized a series of platinum(II) terpyridine complexes, featuring an aminostilbene donor–acceptor framework (10–14) (Fig. 5), for the selective colorimetric recognition of Hg²⁺.¹⁷ The complex with a dithiaazacrown moiety (12 and 13) exhibits a highly sensitive and selective colorimetric response to Hg²⁺ in DMF medium through modulation of the relative strength of ICT and MLCT transitions. Spectrophotometric titration of 14 with Hg(ClO₄)₂ gave a similar response, which excluded any interfering effect of counter ions. Initially, Hg²⁺ was bound to the Pt(II)-centre through a usual Hg(II)–Pt(II) bond formation (K₁ = 1.33 × 10⁴ M⁻¹), which further favoured the ICT process. With excess [Hg²⁺], the second Hg²⁺ was bound to the dithiaazacrown moiety (K₂ = 1.64 × 10³ M⁻¹) and suppressed the ICT process, however a new absorption band for a MLCT process was observed. The weak Pt^II⋯Hg^II metallophilic interaction at lower [Hg²⁺] was proven by ¹H NMR titration. These reagents allowed detection of Hg²⁺ in the micromolar level.

Fig. 5 Structure of the chemosensors 10–14.

Citrate-coated silver nanoparticles (15) (Fig. 6) were synthesized by Wang and co-workers in order to selectively recognize Hg²⁺ in aqueous medium.¹⁸ In presence of 10 μM Hg²⁺ the solution colour of Cit–AgNPs changed (from light yellow to deep yellow), accompanying with a corresponding intensity decrease and a slight blue shift in the absorption maxima from 400 to 397 nm. This phenomenon may be ascribed to the reduction of Hg²⁺ by AgNPs and subsequent deposition of elementary Hg on the surface of AgNPs, yielding amalgam particles. However, in the presence of H₂O₂, the system was able to detect nM level of Hg²⁺ present in the aqueous solution. Moreover, in the presence of H₂O₂, reduction from Hg²⁺ to Hg(0) was more efficient and Hg(0) was deposited on the nanoparticle surface with decrease in the surface charge density and this led to the aggregation and a red-shift in the absorption spectra.

Fig. 6 Proposed mechanism for the Hg²⁺-induced colorimetric response of AgNPs (15) in the presence of H₂O₂.¹⁸

Das and co-workers developed a diametrically disubstituted 1,4,8,11-tetraazacyclotetradecane (cyclam) derivative, functionalized with 4-(4-dimethylamino)phenyl azobenzene as the signalling moiety, which has shown remarkable specificity towards Hg²⁺ (Fig. 7) in CH₃CN–aq. HEPES buffer (2 : 3, v/v; pH 7.2) medium.¹⁹ However, solubility of the reagent 16 could be further improved simply by allowing this to form an inclusion complex ([3]pseudo rotaxane; 16·2β-CD) with β-cyclodextrin (β-CD). This inclusion complex (L·2β-CD) could be used for developing a more intense colour on binding to Hg²⁺ in CH₃CN–HEPES buffer medium. Non-toxic nature of L and L·2β-CD was checked with the living cells of a Gram-negative bacterium (Pseudomonas putida). Experiments revealed that these two reagents could be used as a staining agent for detection of Hg²⁺ present in this microorganism, while the intensity of the stained bacterium cells was more intense for L·2β-CD.

Fig. 7 (A) Diametrically disubstituted cyclam unit with azo-chromophore (16) for the recognition of Hg²⁺; (B) change in absorption spectra of azamacrocyclic derivative (16) as a function of [Hg²⁺], Inset: Least square plot of Δ(A − A₀) vs. [Hg²⁺] at 494 nm; (C) schematic presentation for the formation of L, Hg²⁺·L, L·2β-CD, Hg²⁺·[L·2β-CD]. Reprinted with permission from ref. 19. Copyright 2010 American Chemical Society.

The same group later developed a colorimetric sensor 17 (Fig. 8), which could act as a colorimetric sensor for Hg²⁺ (K_a = 6.54 ± 10⁴ M⁻¹) and Cr³⁺ (K_a = 4.31 ± 10⁴ M⁻¹) among various Group 1A, IIA and all other common transition metal ions in acetonitrile medium.²⁰ On binding of 17 to the Hg²⁺ or Cr³⁺ ions, the absorption band at 440 nm for 17 was found to decrease with a simultaneous growth of an absorption band with maxima at 509 nm, along with an isosbestic point at 464 nm, which was attributed to a charge-transfer transition. A visually detectable change in the solution colour from yellow to red was observed. A test paper kit for the detection of Hg²⁺ or Cr³⁺ in neutral aqueous media was also developed (Fig. 8).

Fig. 8 (A) Structure of the chemosensor 17. (B) The variation in the colour of the test strip for 17 after dipping in aqueous solutions with varying concentrations of Hg²⁺ and Cr³⁺. A solution of 17 with a concentration of 0.002 M was used to develop the strip. Reprinted with permission from ref. 20. Copyright 2012 Royal Society of Chemistry.

Rhodamine-based reversible receptors for the recognition of Hg²⁺ ions

Xanthene-based fluorophores, including rhodamine and fluorescein, are being used extensively for molecular recognition because of their rich spectral properties. These dyes show high extinction coefficients for electronic spectral band at ≥475 nm, strong emission band at ≥550 nm with high quantum yield value and appreciably high photo-stability towards radiation of visible wavelength.²¹ More recently, rhodamine derivatives have received certain importance as a staining agent or as an imaging reagent as these derivatives in their spirolactone or spirolactam form neither show any absorption nor any emission spectral band beyond 380 nm, while in the acyclic xanthene form, they have a strong absorption (∼525 nm) and emission (∼560 nm) bands. These account for a visually detectable change in solution colour from colourless to pink with an associated ‘turn on’ fluorescence response. In general, such changes happen due to a reversible conversion from a cyclic spirolactam form to an acyclic xanthene form. These changes could also be achieved upon the reversible reaction with an appropriate metal ion or H⁺ (Fig. 9).

Fig. 9 Schematic presentation of the change from cyclic lactam form to an acyclic xanthenes form of the rhodamine derivatives on binding to a metal ion.

Utilizing these optical spectral responses and reversible equilibrium that exists between spirolactam/spirolactone and the corresponding xanthene form, several probes have been designed for the recognition of certain metal ions. Possibility of using such probes as biomarkers has been explored in certain instances.

Chang and his co-workers had shown that colourless solution of rhodamine B hydrazine (18), in methanol–aq. acetate buffer (1 : 9, v/v; pH = 5) turned red with associated emission band maximum at 578 nm on binding to Hg²⁺ within 10 min (Fig. 10) of the addition of Hg²⁺ to the reagent solution.²² Studies revealed that 18 underwent a decomposition reaction with Hg²⁺ to regenerate the acyclic xanthene form with turn on absorption and emission responses at about 530 and 560 nm, respectively. Minimum detection limit for Hg²⁺ was found to be 2 μM, while no rate constant was reported.

Fig. 10 Schematic representation of rhodamine B hydrazine hydrolysis with switch on absorption and emission responses at 530 nm and 560 nm, respectively.

Huang et al. recently reported a multi-signalling sensor 19 for the recognition of Hg²⁺ using a rhodamine derivative functionalized with redox active ferrocenyl (Fc) and 8-hydroxyquinoline moieties for probing the binding phenomena either through change(s) in optical spectra or in electrochemical redox potentials (Fig. 11).²³ On selective binding to Hg²⁺ in ethanol–HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer (1 : 1, v/v, pH 7.2), sensor 19 displayed a visually detectable fluorescence on response, along with a change in colour from colourless to pink. Presence of the Fc moiety also helped in monitoring the Hg²⁺·19 formation through changes in the redox potential of Fc/Fc⁺ couple from 0.40 to 0.15 V on binding to Hg²⁺ ion. A moderate formation constant (3.7 × 10³ M⁻¹) for Hg²⁺·19 was observed. Confocal laser scanning microscopic studies further revealed the possibility of using this as an imaging reagent for in vivo detection of Hg²⁺ in living cells.²³

Fig. 11 (A) Schematic presentation for the receptors 19 and 20 with possible binding modes to Hg²⁺.

Qian et al. developed a rhodamine-based sensor (20) bearing ionophore NS₂ (Fig. 11B), which showed high affinity towards Hg²⁺.²⁴ In CH₃CN–HEPES buffer (3 : 17, v/v; pH 6.98) solution 20 was found to bind selectively to Hg²⁺ (1 : 1 binding stoichiometry) with change in colour from colourless to purple with associated fluorescence on response at ∼570 nm. A binding constant of 1.18 × 10⁶ M⁻¹ was evaluated for Hg²⁺·20 formation.

Zheng, Xu, and their co-workers utilized a rhodamine B thiohydrazide derivative 21 (ref. 25) (Fig. 12), as a fluorescent chemosensor for Hg²⁺, which could reversibly bind Hg²⁺ in aqueous solution at pH 3.4 in a highly selective manner. Coordination of Hg²⁺ to the N and S binding sites in 21 with 1 : 2 (Hg²⁺ : 21) stoichiometry and opening of the spirolactam ring was proposed. Zhang and co-workers reported another fluorescent probe, 22 (Fig. 12) in which rhodamine unit was functionalized with coumarin moiety and used for the detection of Hg²⁺.²⁵ Moderately high binding constant (1.18 × 10⁶ M⁻¹) in ethanol–tris–HCl buffer (1 : 1, v/v; pH 7.24) was reported. Binding stoichiometry of 1 : 1 and specificity in binding towards Hg²⁺ in presence of all other competing metal ions was established by detailed spectral studies. Reversibility in the Hg²⁺·22 formation was established using little excess of EDTA as the competing ligand. Reported lower detection limit of 8 ppb was much closer to the concentration permitted by United States environment protection agency (USEPA) for safe drinking water and could be used for the detection of Hg²⁺ in both tap and river water samples.

Fig. 12 Molecular structures of the receptors 21, 22 and 23.

Li and co-workers reported a rhodamine derivative functionalized with a β-napthol moiety and this receptor (23, Fig. 12) showed selective binding to Hg²⁺ over other metal ions in aqueous solution, except Cu²⁺.²⁶ Note that Job’s plot analysis suggested a 1 : 1 binding stoichiometry. Binding process (K_a^Hg2+ = 8.42 × 10⁴ M⁻¹) was probed by monitoring fluorescence enhancements at ∼555 nm. However, fluorescence responses of 23 at 555 nm for a definite sequence of ionic inputs in the form of Hg²⁺ and/or Cu²⁺ ion could be correlated for demonstrating a molecular level keypad lock.

Selective detection of Hg²⁺ in aqueous media was also reported by He and Duan et al. using a set of rhodamine based sensors, 24, 25 and 26 (Fig. 13), having thiophene moiety as one of the coordination modes.²⁷ In case of 24 and 26, interference from Cu²⁺ and Pb²⁺/Ag⁺, respectively, was observed in the detection of Hg²⁺; whereas in the case of 25 no such interference was observed. For 25, a 1 : 2 (Hg²⁺ : 25) binding stoichiometry with a relatively high binding constant (K_a = 8.18 × 10⁷ M⁻²) was observed. Reagent 24 also showed similar binding mode with high association constant (K_a = 1.58 × 10¹³ M⁻²), while 26 showed 1 : 1 binding stoichiometry with moderate affinity constant (4.8 × 10⁶ M⁻¹). Lowest detectable concentration for Hg²⁺ reported as low as ∼1 ppb for 26, which was lower than the limit set by various regulatory agencies (EPA or WHO) for safe drinking water.

Fig. 13 Structure of the chemosensors 24–26.

Colorimetric and fluorescent off–on responses for a thiospirolactam–rhodamine derivative (27) (Fig. 14) were reported for the detection of Hg²⁺ by Xu and his co-workers.²⁸ Moderate association constant for Hg²⁺·{27}₂ (K_a = 5.20 × 10⁵ M⁻²) formation was estimated. This reagent could be used for in vivo imaging of Hg²⁺ present in rat Schwann cells.

Fig. 14 Structure of 27 and the possible binding mode for Hg²⁺.

Recently, Das and his research group have reported a rhodamine 6G derivative (28), which showed changes in the absorption and emission spectral patterns with associated changes in visually detectable solution colour and fluorescence, respectively, on specific binding to Hg²⁺ in CH₃OH : H₂O medium (1 : 1, v/v; pH 7.0) (Fig. 15).²⁹

Fig. 15 Binding mode of Hg²⁺ and Cu²⁺ with 28 in MeOH–water (1 : 1, v/v, pH 7.0). Inset: Optical microscopic images for (A) cells of Pseudomonas putida exposed to Hg²⁺ solution (10 mM) and (B) then subsequently exposed to a water–ethanol (7/3, v/v) solution of 28 (20 μM). Reprinted with permission from ref. 29. Copyright 2008 American Chemical Society.

For Cu²⁺, analogous changes in absorption spectra and solution colour were also observed. However, no new emission band beyond was observed on binding to Cu²⁺ after excitation at ∼530 nm, and this could be attributed to the paramagnetic effect of the unpaired Cu^II-d⁹ electron. Observed binding stoichiometry for Cu²⁺ was 1 : 1, while that for Hg²⁺ was 1 : 2. The receptor 28 was found to be reversible with KI as the solution of 28. Note that Hg²⁺ turned colourless due to formation of HgI₄²⁻. The receptor 28 was further utilised as a staining, as well as an imaging reagent, for the detection of Hg²⁺ uptake in a bacteria for viewing through optical and fluorescence microscopes, respectively.

Duan, Li and their associates have developed a water-soluble rhodamine derivative (Fig. 16) by tagging the hydrophilic glucose unit, and this modified receptor could bind specifically to Hg²⁺ in aqueous medium following a 1 : 1 binding stoichiometry.³⁰

Fig. 16 Molecular structure of the receptors 29 and 30 [inset: (a) cells stained with 100 μM 29 for 10 min at 25 °C, (b) supplemented cells loaded with 10 μM Hg(NO₃)₂, (c) bright-field image and (d) overlay image. Reprinted with permission from ref. 30. Copyright 2009 Royal Society of Chemistry.

The lowest detection limit evaluated for Hg²⁺ detection was 1 ppb. The receptor 29 exhibited a very weak fluorescence at 550 nm, while on binding to Hg²⁺, a significant enhancement in the emission band intensity was observed due to its transformation into the corresponding xanthene form. The association constant determined from the emission titration was (5.4 ± 0.1) × 10⁵ M⁻¹. Reversibility of the binding process was established after treatment with Na₂S or NaI. Confocal laser microscopic studies revealed that this imaging reagent could detect Hg²⁺ uptake in HeLa cells from a solution having [Hg²⁺] as low as 10 μM. The red fluorescence along the periphery of the cells indicated accumulation of Hg²⁺ at the cytosol.

Receptor 30 was reported by Tang, Nandhakumar and their co-workers (Fig. 16).³¹ This reagent showed Cu²⁺-specific enhancement in the intensity of the electronic spectra with maxima at ∼530 nm. Due to paramagnetic coupling, no change in emission was observed on binding to Cu²⁺. In contrast, a significant enhancement in emission intensity at ∼550 nm was observed for Hg²⁺.

Ferrocene containing rhodamine 6G-based multi-responsive chemosensors, 31 and 32, which were also developed by Duan's group for the recognition of Hg²⁺ in pure water medium (Fig. 17).³² As anticipated, absorption and emission spectral data recorded for both receptors showed ‘turn on’ response on binding to Hg²⁺ for the generation of the corresponding acyclic xanthene forms. The emission quantum yield value for reagent 31 was evaluated as 0.38 at 550 nm when bound to Hg²⁺. The Benesi–Hildebrand plot based on ΔI (I is change in emission intensity at 550 nm for 31) from the systematic titration data confirmed the 1 : 1 binding stoichiometry with an association constant of 1.16 (±0.04) × 10⁶ M⁻¹. Presence of ferrocene unit allowed probing the binding process by monitoring the Fc/Fc⁺ potential changes on binding to Hg²⁺. An anodic shift of 50 mV for Fc/Fc⁺ redox couple was observed on binding of 31 to Hg²⁺. For the reagent 32 (Fig. 17), changes were comparatively less, which signified a slightly weaker binding of this reagent to Hg²⁺. B–H plot revealed an association constant of 2.8 ± 0.2 × 10⁵ M⁻¹ and a binding stoichiometry of 1 : 2.

Fig. 17 Multichannel receptors for the recognition of Hg²⁺ in natural water medium.

Cyclen(1,4,7,10-tetraazacyclo decane) could also be used as an additional binding site in a rhodamine derivative for the co-ordination to a metal ion and accordingly, a new receptor 33 was used for studies for binding to Hg²⁺ (Fig. 18).³³ B–H plot revealed a 1 : 2 binding stoichiometry and was also confirmed from the ESI-Ms data. It showed a 1700-fold enhancement in the emission intensity at maxima of 580 nm upon addition of 10 equivalents of Hg²⁺. Association constant was evaluated from the B–H plot and found to be 2.3 × 10⁸ M⁻².

Fig. 18 Molecular structures for receptors 33 [inset: change in solution luminescence of 33 on binding to Hg²⁺ under UV-light]; structure of the chemosensor 34. Reprinted with permission from ref. 33. Copyright 2008 American Chemical Society.

Ghosh and co-workers developed a bis-sulfonamide derivative of rhodamine B (34) for the selective recognition of Hg²⁺ and Cu²⁺ in CH₃CN : H₂O = 4 : 1, v/v (10 mM tris–HCl buffer, pH 6.8) medium (Fig. 18).³⁴ The colourless solution of 34 turned pinkish along with the increase in absorption at 555 nm in presence of both Hg²⁺ and Cu²⁺ ions due to the opening of the spirocyclic rings of rhodamine moieties. Again, on excitation at 510 nm, Cu²⁺ induced strong emission at 524 and 580 nm; however, in case of Hg²⁺ there was ratiometric response, decrease in emission at 524 nm and moderate enhancement of emission at 580 nm. It was stated that 34 showed different emission response towards Hg²⁺ and Cu²⁺ due to the different binding behaviour of the receptor. Binding stoichiometry of 1 : 1 was evaluated for both Cu²⁺ and Hg²⁺ ions towards 34. Binding constant for metal ions was evaluated from emission titration results ((9.05 ± 0.63) × 10⁴ M⁻¹ for Cu²⁺ and (7.87 ± 0.55) × 10⁴ M⁻¹ for Hg²⁺). The reagent was also used as an imaging reagent for the detection of these ions in human cervical cancer (HeLa) cells.

Boronic acid based receptors 35 and 36 were reported by Yoon and his co-workers (Fig. 19).³⁵ Both reagents showed ‘on–off’ type response on binding to Hg²⁺ in acetonitrile–aq. HEPES buffer medium (9 : 1, v/v), following a 1 : 1 stoichiometry. The association constants of 3.3 × 10³ M⁻¹ and 2.1 × 10⁴ M⁻¹ were reported for reagents 35 and 36, respectively. Presence of additional boronic acid moiety at the binding site could have contributed to the higher association constant of receptor 36.

Fig. 19 Boronic acid containing rhodamine B derivatives for the recognition of Hg²⁺ in aqueous environment.

Das and co-workers developed a new rhodamine-based receptor (37; Fig. 20) that was functionalized with an additional fluorophore (quinoline), which could selectively recognize Hg²⁺ with an interference of Cr³⁺ in an acetonitrile–HEPES buffer medium of pH 7.3.³⁶ 37 could be used as a dual probe and allowed detection of these two ions by probing changes in absorption and the fluorescence spectral pattern. In both instances, the extent of the changes was significant enough to allow visual detection. Interestingly, quinoline-based emission was not observed either in 37 or in 37·Hg²⁺ owing to C=N isomerization and intersystem crossing during the recognition of Hg²⁺ ions, respectively.

Fig. 20 Receptor 37 showed spirolactam ring opening phenomenon with ionic Hg(II) salts but could not show similar property with covalent Hg(II) salts [inset: dark-field image of MCF7 cells with 37 (4.0 μM) and ionic Hg²⁺ (2.0 μM)]. Reprinted with permission from ref. 36. Copyright 2012 American Chemical Society.

More importantly, the receptor molecule could be used as an imaging reagent for detection of Hg²⁺ uptake in live human cancer cells (MCF7) using laser confocal microscopic studies. Unlike Hg(ClO₄)₂ or Hg(NO₃)₂ salts, HgCl₂ or HgI₂ failed to induce any visually detectable change in colour or fluorescence upon interaction with 37 under identical experimental conditions. Presumably, the higher covalent nature of Hg(II) in HgCl₂ or HgI₂ accounts for its lower acidity, and its inability to open up the spirolactam ring of the reagent 37. The issue had been addressed on the basis of the single-crystal X-ray structures of 37·HgX₂ (X⁻ = Cl⁻ or I⁻) and results from other spectral studies. Thus, this article also revealed the role of the metal ion acidity in effectively opening the spirolactam ring of the rhodamine derivatives.

A new tripodal receptor 38 (Fig. 21) was reported by Ghosh and co-workers, which could selectively recognize Hg²⁺ ions in CH₃CN–water (4 : 1, v/v; 10 μM tris–HCl buffer, pH 7.0) medium.³⁷ Upon gradual addition of Hg²⁺, emission at 536 nm was quenched and a new emission was observed at 580 nm. This ratiometric response on binding to the Hg²⁺ ion was attributed to the slightly different binding mode of reagent 38 (Fig. 21). A binding stoichiometry of 1 : 1 was proposed with binding constant of 6.31 ± 0.74 × 10⁵ M⁻¹ between 38 and Hg²⁺. Furthermore, the receptor showed in vitro detection of Hg²⁺ ions in human cervical cancer (HeLa) cells. One major limitation of this reagent was the non-specific binding to most other transition metal ions. However, binding constants for such metal ions were lower by an order of magnitude as compared to that for Hg²⁺.

Fig. 21 Structure of 38 and the possible binding modes for Hg²⁺.

Li et al. introduced hydrazinobenzothiazole unit in rhodamine systems 39 (Fig. 22A) for the development of first six-membered spirocycle ring for the recognition of Hg²⁺ in ethanol–PBS buffer (4 : 6, pH 7.4).³⁸ A change in emission enhancement of 1000-fold with the quantum yield of 0.87 was observed for Hg²⁺, while the reported lower detection limit was 30 nM. The observed association constant was 1.02 × 10⁶ M⁻¹ with binding stoichiometry 1 : 1; however, receptor 39 showed some affinity towards Ag⁺ ions under the experiment condition. Probe 39 was further used to the map the sorption of Hg²⁺ to bacteria-EPS-mineral aggregates under anoxic condition and adsorption of Hg²⁺ on the cell surface was confirmed from the overlay of fluorescence and reflection image.

Fig. 22 (A) Structure of six-membered spirocycle containing rhodamine 39 for Hg²⁺ recognition. (B) Structure of the rhodamine derivatives 40, 41 and 42.

Bhattacharya and co-workers developed rhodamine based positional isomers (40, 41 and 42; Fig. 22B) for the detection of metal ions in aqueous medium.³⁹ The chemosensor 40, with pyridine nitrogen at o-position, showed a selective response towards Cu(II), whereas 41 and 42 with pyridine nitrogen at m- and p-position, respectively, could show specificity towards Hg²⁺ among various cations tested. The colourless solution of 41 and 42 became pink in presence of Hg²⁺ typically with the appearance of new absorbance maxima at 530 nm due to the opening of the spirocyclic ring of these rhodamine derivatives, while the associated emission maxima appeared at 560 nm (λ_ex = 520 nm). A 1 : 1 binding stoichiometry was evaluated for binding with Hg²⁺ for both 41 (log K = 3.7) and 42 (log K = 3.91). Lowest detection limits reported for Hg²⁺ for receptors 41 and 42 were 9.4 and 4 ppb, respectively. Furthermore, the sensitivity of Hg²⁺ ion recognition by probes 41 and 42 in the presence of excess of plasma protein (BSA) and human blood serum was also investigated.

Among different non-radiative energy transfer processes between two fluorophores, resonance energy transfer (RET) is the most common and important one. RET is a distance-dependent physical process, by which energy is transferred non-radiatively from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor) by means of intermolecular long-range dipole–dipole coupling. The use of the RET process for the design of a molecular probe is generally preferred, as this helps in overcoming the problems posed by phenomena like photo bleaching and aggregated self-quenching.

Receptor 43 is a good demonstration of a RET-based reagent, in which resonance energy had been transferred from dansyl to xanthene form of the rhodamine moiety on binding to a Hg²⁺ in CH₃CN–water medium (Fig. 23).⁴⁰ The RET was confirmed from the rhodamine-based emission observed at 555 nm on excitation at the absorption maxima (λ_ext 340 nm) of the dansyl unit. The singlet–singlet energy transfer efficiency (Φ_ET) and the rate constant for the energy transfer process from dansyl to ring-opened rhodamine acceptor was 83% and 2.84 × 10⁸ s⁻¹, respectively. A moderate association constant of (5.0 ± 0.2) × 10⁴ M⁻¹ was evaluated for Hg²⁺, while this reagent showed a very weak interference from Cu²⁺. Receptor 43 could detect Hg²⁺ as low as 0.1 ppm level and also could be used as a staining agent for detection of uptake Hg²⁺ in Pseudomonas putida bacteria.

Fig. 23 Resonance energy transferring probe during the recognition of Hg²⁺ in acetonitrile–water medium (1 : 1, v/v) [inset: use of receptor 43 for the recognition of Hg²⁺ within the Pseudomonas putida bacteria viewed under confocal laser microscopy; (a) only 43 in the cells, (b) 43 in presence of Hg²⁺ within the cells]. Reprinted with permission from ref. 40. Copyright 2009 American Chemical Society.

FRET process was also effectively used by Zeng and co-workers who designed another ratiometric sensor, i.e., 44 (Fig. 24), for detection of Hg²⁺ ion.⁴¹ FRET process thus achieved was a nice demonstration of self-assembly process that brought the donor and acceptor moieties together. Adamantyl group in aqueous medium is known to form inclusion complex with β-CD and this was used to assemble the donor–acceptor units. On addition of Hg²⁺, ring-opening process took place and on excitation of the donor at 495 nm (with λ_Ems = 518 nm) FRET process became effective with acceptor Hg²⁺-bound rhodamine based emission at 586 nm appeared. The detection limit for Hg²⁺ was determined to be 10 nM, while binding constant of 5.3 × 10⁷ M⁻¹ was evaluated for 1 : 1 binding stoichiometry. Both untreated HeLa and L929 cells, as well as pre-exposed (and washed) with Hg²⁺ were subsequently stained with 44, while a fluorescence change from green for untreated to red for cells pre-exposed to Hg²⁺ was observed. This confirmed that the FRET process was operational even in live cells.

Fig. 24 (a) Structure of the chemosensor 44; (b) Schematic for the FRET-based ratiometric sensing system for Hg²⁺ with β-CD as vehicle; (c) Fluorescence microscope imaging of HeLa and L929 cells stained with CD-based sensor before (A and C) and after (B and D) these were pre-exposed to 1 ppm of Hg²⁺. Reprinted with permission from ref. 41. Copyright 2010 American Chemical Society.

A calix[4]arene derivative-based chemosensor (45) was reported by Kim et al. (Fig. 25).⁴² A strong spectral overlap between pyrene excimer emission band and rhodamine ring-opened absorption band was observed and initiated a FRET-based response on selective binding to Hg²⁺ ion with an enhancement of emission at 576 nm (λ_ext = 343 nm).

Fig. 25 Structure of the chemosensor 45 and Hg²⁺-induced FRET response.

More recently, Das and co-workers developed new rhodamine-based chemosensors, i.e., 46 and 47 (Fig. 26), which were found to show preferences towards Hg²⁺ and Cr³⁺ in presence of large excess of all other competing transition metal ions.⁴³ Specific binding of this reagent either to Hg²⁺ or Cr³⁺ resulted associated changes in their optical and fluorescence spectral behaviour, which were significant enough to allow their visual detection. For the reagent 46, the lower detection limit was even lower than the permissible [Hg²⁺] or [Cr³⁺] in safe drinking water as per the standard U.S. EPA norms.

Fig. 26 Structure of the chemosensors 46, 47 and 48.

The other receptor 47 could be used as a ratiometric sensor for detection of Hg²⁺ and Cr³⁺ based on the resonance energy transfer (RET) process, involving the donor naphthalimide and the acceptor Hg²⁺/Cr³⁺-bound xanthene fragment. Furthermore, confocal laser microscopic studies confirmed that the reagent 47 could also be used as an imaging probe for detection of uptake of these ions in A431 cells. Reagent 46 was actually synthesized to establish the binding mode, while results of reagents 46 and 47 were compared to establish the FRET-based luminescence responses on binding to Hg²⁺ or Cr³⁺. Authors could also demonstrate that use of an appropriate masking agent (KI) allowed them to evaluate the concentration of individual metal ions in the solution that have these two ions. Higher affinity of I⁻ towards Hg²⁺ led to the formation of HgI₄²⁻ and allowed the binding of Cr³⁺ to these set of reagents.

Another well-known energy transfer mechanism, i.e., bond energy transfer (TBET), was also utilized for the recognition of Hg²⁺ ions. Unlike RET process, TBET process does not require the spectral overlap between donor emission and acceptor absorption bands and often lead to large pseudo-Stokes shifts (∼200 nm) within rigid systems. Most recently, Kumar et al. established a TBET-based chemosensor, 48, by combining naphthalimide and rhodamine through a conjugated spacer.⁴⁴ Upon addition of Hg²⁺ to the THF–H₂O (9.5 : 0.5, v/v) medium of 48 (Fig. 26), a new absorption band appeared at 565 nm along with a colour change from colourless to pink, and an emission band characteristic of ring-opened rhodamine also appeared at 578 nm when the chemosensor was excited at 360 nm. Note that a detection limit of 2 × 10⁻⁶ M for Hg²⁺ and a 1 : 1 stoichiometry for the binding mode with a binding constant of 7.1 × 10⁴ M⁻¹ were evaluated. The authors described 48 as a potential fluorescence imaging agent for the detection of Hg²⁺ in live prostate cancer (PC3) cells.

The same group have recently developed a pentaquinone–rhodamine conjugate 49 for the recognition of Hg²⁺ ions in THF–water medium (9.5 : 0.5, v/v) (Fig. 27A).⁴⁵ The UV-vis spectrum of 49 exhibits absorption bands at 275 nm and 322 nm in THF–H₂O due to electronic transitions associated with pentaquinone moiety.

Fig. 27 (A) Rhodamine-based reversible receptor for the recognition of Hg²⁺ showing TBET mechanism [inset: PC3 cell lines (a) with receptor 49 (1.0 μM), (b) in presence of Hg²⁺ (30.0 μM)]. Reprinted with permission from ref. 45. Copyright 2012 American Chemical Society. (B) Structure of the chemosensor 50.

However, upon the addition of Hg²⁺ ions (0–200 mole equivalent), the intensity of these absorption bands was found to increase; however, along with these increases, a new absorption appeared at 554 nm and the solution colour turned pink. Furthermore, on excitation at 360 nm, an intense emission band at 582 nm was observed in presence of Hg²⁺ due to the transfer of resonance energy from pentaquinone to rhodamine via through-bond pathway. TBET mechanism was established based on studies with relevant model systems. This reagent could even be used as an imaging reagent for the detection of Hg²⁺ in PC3 cell lines by confocal laser microscopy. Lowest [Hg²⁺] that this reagent could detect was found to be 0.14 ppb, which is lower that the safe limit for [Hg²⁺] for safe drinking water.

Das et al. reported a unique example of interrupted PET-coupled TBET phenomena for the selective recognition of Hg²⁺ in MeOH–aq. HEPES buffer (1 : 1, v/v) (pH 7.2) using a new coumarin–rhodamine conjugate 50 (Fig. 27B).⁴⁶ It showed simultaneous enhancement of coumarin-based emission at 402 nm and rhodamine-based emission at 585 nm (λ_ext = 320 nm) upon Hg²⁺ binding. Binding of the Hg²⁺ to the pendant amine moiety (coupled to coumarin fragment) resulted an interruption of PET process and a switched on luminescence response from the coumarin fragment, which in turn initiated a TBET process from Hg²⁺-bound coumarin moiety to the acceptor Hg²⁺-bound rhodamine fragment in Hg²⁺·50. The photophysical phenomenon was rationalized based on the luminescence responses of two appropriate model compounds under identical experimental conditions. Benesi–Hildebrand plot of the emission titration results established a 1 : 1 stoichiometry with an association constant of 7.3 (±0.6) × 10⁴ M⁻¹ between 50 and Hg²⁺. Furthermore, 50 could be used as an imaging reagent for determining intracellular distribution of Hg²⁺ in MCF7 cells exposed to [Hg²⁺] as low as 2 ppb.

Bhalla and Kumar et al. developed two hexaphenylbenzene (HPB) derivatives (51 and 52) having rhodamine B moiety for the selective recognition of Hg²⁺ in both polar aprotic (CH₃CN and THF) and protic medium (MeOH) (Fig. 28).⁴⁷ For both reagents, binding to Hg²⁺ ion resulted in opening of the spirolactam ring with a new strong absorption band that had a distinct spectral overlap with the emission band of the HPB moiety (λ_ext = 290 nm). This accounted for a nonradiative energy transfer from donor HPB moiety to the Hg²⁺ bound rhodamine moiety as the acceptor, which resulted in a strong rhodamine-based luminescence band with maxima at 585 nm. This resulted in a large pseudo-Stokes shift of 295 nm. The energy transfer efficiency was better in CH₃CN than THF medium for both 51 and 52. CH₃CN being a better coordinating solvent, reduced the efficiency of the complexation of Hg²⁺ ion with the rhodamine based receptor. Furthermore, these receptors showed through bond energy transfer (TBET) in polar protic solvent MeOH. Receptor 51 showed higher through bond energy transfer efficiency compared to 52 in the presence of Hg²⁺ ions. Higher conjugation between the donor and acceptor moieties in 52 accounted for the weaker TBET efficiency in comparison to 51.

Fig. 28 Structures of the chemosensors 51 and 52.

Fluorogenic receptors for Hg²⁺ other than rhodamine

Highly fluorescent 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (better known as BODIPY, difluoroborondipyrromethene)⁴⁸ derivatives are one of the most popular dye molecules that are being used in biological labelling and electroluminescent devices. It has uses as a fluorescence-based signalling moiety for recognition studies for their rich photo-physical properties. The BODIPY derivatives show relatively high molar absorption coefficient (ε), emission quantum yield (Φ) and negligible triplet state formation. Excitation/emission wavelengths for these derivatives generally appear in the visible region and, more importantly, this class of compounds generally have fairly stable excited states (τ in the nano-second time range). Apart from these, synthetic accessibility of various derivatives, good solubility in common solvents, resistance towards self-aggregation in solution have actually made this class of molecule a popular choice for the researchers.

BODIPY fluorophore with the binding site azadioxadithia-15-crown-5-chelator could effectively recognize Hg²⁺ ions. Thus, receptor 53 was designed with two BODIPY units: one as donor and another as an acceptor, while thio-crown ether moiety acted as the receptor fragment for Hg²⁺ ions (Fig. 29).⁴⁹ The binding of Hg²⁺ caused blue shifts in the absorption spectrum of the longer wavelength dye. This fact increased the spectral overlap between donor emission and acceptor absorption, and thus the FRET response. As anticipated, a 1 : 1 complexation with Hg²⁺ with the dissociation constant of 4.5 × 10⁻⁷ M was evaluated in THF medium. Analogous azadioxadithia crown derivative containing receptor 54 in BODIPY system was reported by Zhu et al.⁵⁰ for the recognition of Hg²⁺ (Fig. 29). Receptor absorbed at 606 nm while its emission was at 668 nm with very low quantum yield. Addition of Hg²⁺ resulted disappearance of the band at 660 nm with concomitant growth of the band at 564 nm with change in colour from purple to pink-red. On the other hand, generation of a new emission band at 578 nm with 7-fold enhancement in emission intensity was observed. These spectral changes were ideally suited for ratiometric fluorescent detection. The coordination of Hg²⁺ to the macrocyclic moiety did not favour the ICT process, and this resulted in the observed blue shifts in optical spectra.

Fig. 29 Chemosensors with azadioxadithia-15-crown-5 as a cheater for Hg²⁺ ion recognition.

Another similar BODIPY based receptor, 55, was developed for the recognition of Hg²⁺ ions in THF medium (Fig. 30).⁵¹ Titration of 55 was performed with Hg²⁺ revealed that the two overlapping absorption spectral bands got resolved as ICT process was interrupted due to coordination of Hg²⁺ to the aza-crown ether moiety. Emission intensity at 675 nm was found to increase gradually on increasing the [Hg²⁺]. The Benesi–Hildebrand plot revealed the 1 : 1 binding stoichiometry with the association constant of 7.8 × 10⁵ M⁻¹.

Fig. 30 Receptor 55 for the recognition of Hg²⁺ in THF medium [inset: change in colouration in presence of Hg²⁺ on excitation with UV-lamp of wavelength 354 nm and relative intensity changed for Hg²⁺ ions compared to other ions]. Reprinted with permission from ref. 51. Copyright 2009 American Chemical Society.

Receptor 56 was synthesized from BODIPY and porphyrin, which showed an appreciable spectral overlap between two dyes with larger pseudo-Stokes shifts (Fig. 31A).⁵² Moreover, the FRET process helped in nullifying the back-scattering problem. 8-Hydroxyquinoline benzoate moiety acted as the binding site and also favoured the energy transfer process during the recognition of Hg²⁺ and Fe²⁺ ions. Absorption band at 506 nm was assigned to a BODIPY-based CT transition, while bands at 420 nm, 550 nm, 591 nm and 648 nm were attributed to porphyrin-based transitions. A comparison of the spectra of 56 with those of individual components revealed absence of any ground state interaction. Energy transfer efficiency was found to be 95.4% on binding to Hg²⁺. Binding stoichiometry of 1 : 1 and an association constant of 3.5 × 10⁶ M⁻¹ in DMF–MeOH medium (98 : 2) were reported.

Fig. 31 (A) BODIPY porphyrin-based versatile fluorescence resonance energy transfer (FRET) ratiometric sensor for Hg²⁺. (B) Pyrene-based receptor for Hg²⁺ recognition with excimer formation. Inset: Emission spectra recorded with different metal ions. Reprinted with permission from ref. 53. Copyright 2011 American Chemical Society.

Receptor 57 showed two emission bands at 383 nm and 402 nm corresponds to an individual pyrene emission, while addition of Hg²⁺ led to the growth of new emission band at 480 nm with lowering the monomer-based emissions with an isoemissive point at 443 nm (Fig. 31B).⁵³ These emission responses at 383 and 480 nm were found to be ratiometric and could be used for the recognition of Hg²⁺ in 10 mM aq. HEPES buffer–DMF (98 : 2, v/v) solution of pH 7.4.

Histidine has affinity for Cu²⁺ ions but its selectivity could be changed by introducing the bis-dithiocarbamate moiety, and accordingly receptor 58 was designed (Fig. 32).⁵⁴ Receptor 58 showed significant enhancement in emission intensity with 50 nm blue shift (from 590 nm to 540 nm) in the emission maxima in presence of Hg²⁺ in CH₃OH–H₂O (80 : 20, v/v with 1% acetonitrile as co-solvent, pH = 7.0) medium with associated change in visually detectable solution fluorescence from red to green on irradiation with 354 nm lamp. The enhancement of Quantum yield (Φ) value was found to increase from 0.022 to 0.402 at 540 nm. The binding stoichiometry and association constant was evaluated as 1 : 1 and 1.25 (±0.4) × 10⁶ M⁻¹, respectively. Authors could even prepare a paper strip, which could be used for the detection of Hg²⁺ in aqueous medium. Moreover, this could be used as an imaging reagent for detection of the Hg²⁺ uptake in HeLa cells, as well as in zebrafish, exposed to a solution having [Hg²⁺] of 10 μM.

Fig. 32 Binding mode of Hg²⁺ in bipodal system [inset: (a) zebrafish with 10 μM of 58, (b) with 10 μM of 58 and 10 μM of Hg²⁺; (c) photographs of the paper strip based sensing with HgNO₃ solutions (left to right): Hg²⁺, 10 μM, 100 μM, and 1 mM Hg²⁺ in aqueous medium buffered at pH 7.0 with HEPES (1 mM) at 25 °C; (d) HeLa cells in presence of 10 μM Hg²⁺ viewed under confocal microscopy]. Reprinted with permission from ref. 54. Copyright 2012 American Chemical Society.

Quinoline-based receptor having azine groups also showed ability to detect Hg²⁺ in THF–aq. phosphate buffer medium (6 : 4, v/v; pH = 7.2).⁵⁵ The receptor 59 showed two main absorption bands at 265 nm and 330 nm, which were attributed to the ¹A to ¹L_a and ¹A to ¹L_b transitions (Fig. 33). Electronic spectral studies revealed that on gradual addition of Hg²⁺, the band at 330 nm was bleached and a new band at 370 nm was developed. Note that receptor 59 showed only a weak emission at 406 nm.

Fig. 33 Detection of Hg²⁺ with azine-based receptors [inset: (a) KB cells, (b) KB cells with 59 (10 μM) and (c) 40 μM Hg²⁺. Reprinted with permission from ref. 55. Copyright 2010 American Chemical Society.

The initial weak emission band at 406 nm was enhanced on binding to Hg²⁺ ion. The restriction of C=N isomerization, and thereby the restricted conformational flexibility due to coordination to Hg²⁺ was accounted for this fluorescence enhancement and an almost switch on response. The binding stoichiometry of 1 : 2 was confirmed from Job's plot as well as from X-ray crystallography. Receptor 59 could further be used for the recognition of Hg²⁺ in live HeLa cells when viewed under confocal microscopy.

Analogous imine-based receptor 60 was also used for specific recognition of Hg²⁺ with ‘turn-on’ fluorescence response (Fig. 34).⁵⁶ Receptor 60 showed three bands in absorption spectra at 255 nm, 295 nm and 385 nm, of which first two were assigned to the naphthalene- and quinolone-based inter-component charge transfer bands, respectively, and the remaining band at longer wavelength was attributed to a CT transition, involving naphthalene donor and quinoline acceptor in THF–phosphate buffer (6 : 4, pH 7.4) solution. Solution fluorescence was found to be very weak after excitation at either 255 nm or at 295 nm. PET process involving the lone pair of electrons of nitrogen quenched the quinoline-based emission. Coordination to Hg²⁺ led to a ‘turn on’ emission response due to the interrupted PET process and the restricted imine rotation. Reversibility in binding of Hg²⁺ to 60 was established by the follow-up treatment with KI. This reagent also could be used for the detection of Hg²⁺ uptake in HeLa cells exposed to a solution having 20 μM of [Hg²⁺].

Fig. 34 Restricted imine isomerisation showing turn on response in emission in presence of Hg²⁺ of the compound 60 [inset: bright-field transmission of living HeLa cells incubated with (a) 60 (20 μM) and (b) 60 (20 μM) with Hg²⁺ (10 μM); (c) fluorescence image of HeLa cells incubated with 60 (20 μM) with Hg²⁺ (10 μM); (d) merged image of (b) and (c)]. Reprinted with permission from ref. 56. Copyright 2012 American Chemical Society.

Chellappa and co-workers developed a new pyrene-based ‘turn on’ fluorescent chemosensor 61 (Fig. 35), which showed significantly enhanced fluorescence intensity on specific binding to Hg²⁺ ions in aq. acetonitrile (30 : 70, v/v) medium.⁵⁷ Binding of Hg²⁺ to 61 caused an interruption in the PET process involving imino nitrogen as well as from the restriction in C=N isomerism. An intermolecular excimer formation resulted in a strong excimer emission at 462 nm (λ_ext = 365 nm). Job's plot and mass spectral analysis confirmed the 1 : 1 complex formation with an association constant of (1.02 ± 0.102) × 10⁴ M⁻¹. Furthermore, 61 could also be used as an imaging reagent for detection of Hg²⁺ present in live Candida albicans cells.

Fig. 35 Structure of the chemosensor 61. Confocal microscopic images of Candida albicans cells incubated with (a) 61 for 30 min, (b) 61 and 10 mM of Hg²⁺ and (c) a bright-field image of probe-treated Candida albicans cells. Reprinted with permission from ref. 57. Copyright 2012 Royal Society of Chemistry.

A unique symmetric fluorescent turn-on and ratiometric chemosensor 62 (Fig. 36) was synthesized by Lee and co-workers from cystine dimer, having two dansyl groups, for the selective detection of Hg²⁺ in 100% aqueous medium.⁵⁸ Upon increase in [Hg²⁺], a 34 nm blue shift in the emission maxima of 62 in HEPES buffer solution (pH = 7.4) was observed with a 3-fold enhancement in emission intensity. A ratiometric response towards Hg²⁺ with a significant increase in the emission intensity around 507 nm and decrease at 583 nm was observed. A 1 : 1 stoichiometry was established from Job's plot analysis and a high dissociation constant of 4.12 × 10⁻⁸ M was obtained from the non-linear least square fitting of the emission titration results. This reagent was also found to be sensitive and the lower detection limit (10 nM) for Hg²⁺ was much lower than the permissible level as per the EPA norms for safe drinking water.

Fig. 36 Structures of the chemosensors 62, 63 and 64.

The same research group developed another dansylsulphonamide-based fluorescent sensor 63, bearing a triazole group using solid phase synthesis and click chemistry (Fig. 36).⁵⁹ Aqueous solution of 63 showed emission at 530 nm upon excitation at 330 nm, which was blue-shifted to 530 nm with a decrease in emission intensity in presence of Hg²⁺ ions. Among various ions tested it showed slight decrease in emission intensity only in presence of Cu²⁺ due to the interaction between the triazole unit and Cu²⁺ ion. The 1 : 1 complexation between the 63 and Hg²⁺ was confirmed by Job's plot and EMI-MS studies. The dissociation constant for Hg²⁺ in aqueous solution was calculated to be 672 nM by the non-linear curve fitting procedure using the emission titration results. ¹H NMR and UV-Vis spectral analysis revealed that two sulfonamide and triazole groups of 63 played a critical role in the interactions with Hg²⁺. The sensor showed Hg²⁺ detection limit of 96 nM in aqueous medium. Furthermore, 63 could penetrate live cells and able to detect the intracellular Hg²⁺ ions confirmed by confocal fluorescence imaging technique.

Todd and Rutledge et al. reported a cyclam-based fluorescent sensor with an appended triazole moiety having a coumarin unit 64 (Fig. 36).⁶⁰ This reagent showed preference towards Hg²⁺ and Cu²⁺ in neutral aqueous solution.⁶⁰ Upon binding of Cu²⁺ and Hg²⁺ ions to the cyclam unit, the coumarin-based emission of 64 was quenched, which accounted for the paramagnetic and the effective spin–orbit coupling, respectively. Fluorescence titrations of 64 with Cu²⁺ and Hg²⁺ confirmed the anticipated 1 : 1 binding stoichiometry, while association constants of 2.2 ± (0.3) × 10⁸ and 7.1 ± (0.8) × 10⁷ M⁻¹ were estimated for Cu²⁺ and Hg²⁺, respectively. The addition of specific anions such as I⁻ and S₂O₃²⁻ causes a complete revival of fluorescence only in the case of Hg²⁺, providing a simple and effective method for distinguishing solutions containing Cu²⁺, Hg²⁺ or a mixture of both ions, even in doped seawater samples. X-ray crystal structures confirmed that pendant triazole coordination occurred through the central nitrogen atom. Fluorescence, mass spectrometry and ¹H NMR experiments revealed that the mechanism of anion-induced fluorescence revival involved either displacement of pendant coordination or complete removal of the Hg²⁺ from the macrocycle, depending on the anion.

Considering the fact that in-field Hg²⁺ detection is crucial for screening of environmental and industrial samples, Zaccheroni et al. developed three new coumarin-based fluorescent chemosensors (65–67) having mixed thia/aza macrocyclic framework as receptors unit (Fig. 37).⁶¹ These probes revealed a fluorescence on response on specific binding to Hg²⁺ in MeCN–H₂O 4 : 1 (v/v) medium. This reagent was further used as an in vitro imaging reagent for detection of cellular uptake of Hg²⁺ ions in Cos-7 (Fig. 37). More importantly, when included in silica core–polyethylene glycol (PEG) shell nanoparticles or supported on polyvinyl chloride (PVC)-based polymeric membranes, ligands (65–67) could also selectively sense Hg²⁺ ions in pure neutral water sample. They also developed an optical sensing array taking advantage of the fluorescent properties of ligand 67 based on the computer screen photo-assisted technique (CSPT). The obtained results showed that by using membranes prepared with ligand 67, they could determine the Hg²⁺ content in water samples in concentrations that are well below the maximal presence recommended by WHO and EPA for safe drinking water.

Fig. 37 Chemical structure of the fluorescent probes (65–67) and (A) Confocal visualization of a Cos-7 cell culture pre-incubated with ligand 67 and (B) pre-incubated with Hg²⁺ ions and successively treated with ligand 67. Reprinted with permission from ref. 61. Copyright 2013 Wiley.

In an effort to develop selective fluorometric chemosensor for Hg²⁺, Bandyopadhyay and co-workers developed a family of A₂B corroles 68, 69, 70 and 71 (Fig. 38).⁶² In toluene medium, they showed a strong emission in the range of 685–705 nm, and among them 71 showed the highest emission quantum yield. Upon addition of Hg²⁺, the greenish solution of 71 turned to yellowish-brown along with a blue shift of ∼27 nm in the UV-Vis spectrum.

Fig. 38 Molecular structures of the chemosensors 68, 69, 70 and 71.

This blue shift was proposed to be due to an interruption in the ICT transitions. However, all these reagents showed fluorescence off response on binding to Hg²⁺ ion, and an efficient spin–orbit coupling process for Hg²⁺ was proposed to be the reason for such a response. A 1 : 1 complex formation with an association constant in the order of 10⁵ M⁻¹ between 71 and Hg²⁺ was evaluated from UV-vis spectral titration. However emission titration provides contradictory result, a 3 : 1 stoichiometry and an overall association constant of 4.57 × 10¹⁵ M⁻¹ between the metal and ligand. To resolve this discrepancy, author performed Stern–Volmer quenching studies using varying [Hg²⁺] and the plot was linear until one mole equivalent of Hg²⁺ was added. Beyond that [Hg²⁺], the plot was exponential, which suggested a static quenching mechanism for lower [Hg²⁺], while for higher [Hg²⁺], a dynamic quenching mechanism was operational due to exciplex formation between the excited corrole monomer and mercury ions.

Ng and co-workers developed a NIR emissive distyryl boron dipyrromethene (BODIPY)-based chemosensor (72) with two bis(1,2,3-triazole)amino substituents and used those for the recognition studies of cations (Fig. 39).⁶³ Among various cations, it gave selective colour and emission change with only Hg²⁺ and Cu²⁺ in CH₃CN–H₂O (1 : 1 v/v) medium. Q band at 686 nm for the absorption spectra of 72 was significantly blue-shifted by 36 or 81 nm on binding to Hg²⁺ or Cu²⁺ ion. Accordingly, the colour of the solution changed from green to pale blue (for Hg²⁺) or deep blue (for Cu²⁺).

Fig. 39 Structures of the chemosensors 72, 73 and 74.

Furthermore, the emission maxima of 72 at 726 nm underwent 40 and 82 nm blue shift on binding to Hg²⁺ and Cu²⁺, respectively. This observed blue shift in both absorption and emission spectra was ascribed to the interruption of ICT upon metal ion coordination with the electron-rich donor moiety of 72. Both Job's plot and emission titration data analysis confirmed the 1 : 2 binding between the host and guest. The association constants calculated from the absorption titrations using least square fitting analysis were (6.2 ± 0.6) × 10⁹ M⁻² for Cu²⁺ and (1.1 ± 0.6) × 10⁹ M⁻² for Hg²⁺.

In an effort to develop a highly selective colorimetric and fluorescent sensor for Hg²⁺, Tong and co-workers synthesized a 2-substituted quinazoline-4(3H)-thione derivative 73 (Fig. 39).⁶⁴ In THF medium, 73 showed quinazoline-based π–π* transition at 366 nm. However, on binding to Hg²⁺, a new MLCT band appeared at 468 nm with an associated colour change from light yellow to dark yellow. Again quinazoline-based emission at 468 nm (λ_ext = 370 nm) was found to quench gradually with the increase in [Hg²⁺]. Job's plot confirmed 2 : 1 adduct formation between 73 and Hg²⁺, while the association constant was evaluated from a nonlinear curve fitting as 4.17 × 10⁸ M⁻². Reversibility of the binding to Hg²⁺ was also established; however, usability of this reagent in non-aqueous medium restricted its usability as an imaging reagent.

Ramesh and Das groups reported a pyrene-based amphiphilic compound 74 (Fig. 39) for the selective recognition of Hg²⁺ in aqueous medium over a broad pH range.⁶⁵ Upon excitation at 340 nm, 74 showed two closely spaced monomer emission bands at around 376 and 394 nm along with a broad structureless band at 516 nm, due to the intermolecular excimer formation. Excitation studies confirmed that the observed excimer formation was static in nature. On formation of a 1 : 1 complex with Hg²⁺, the excimer emission was quenched and monomer emission was enhanced due to the decrease in the extent of intermolecular aggregation of ligand 74. The detection limit of 74 for Hg²⁺ was found to be 8 × 10⁻⁹ M, and the binding constant measured from fluorescence titration was found to be 1.12 × 10⁵ M⁻¹. This reagent was used for detection of Hg²⁺ ion uptake in live human cancer cells (HeLa). Again, the hydrophobic part of 74 was efficiently used for the quantitative extraction of Hg²⁺ from an aqueous medium into the organic layer with an efficiency of about 99%.

Bis-heteroleptic Ru(II) complexes could also be useful for the recognition of the toxic metal ion Hg²⁺ in an aqueous-organic environment (Fig. 40A).⁶⁶ Complex 75 with two benzothiazole amide units of bipyridyl ligand showed 24-fold enhancement by shifting of the emission wavelength from 648 nm to 656 nm with the association constant log β = 7.8 ± 0.3 with a binding stoichiometry of 2 : 1 for Hg²⁺ : 75 in 0.1 M HEPES buffer–CH₃CN (v/v, 1 : 1; pH 7.4).⁶⁶ Moreover, this reversible sensor 75 could detect Hg²⁺ as low as 118 nM level. Interestingly, complex 75 could also recognize Ag⁺ ions under the experimental condition but at different emission wavelength, i.e., 630 nm; thus, it could be useful for the dual-ion sensors.

Fig. 40 (A) Bis-heteroleptic Ru(II)-complex (75) for the recognition of Hg²⁺ ions. (B) Hg²⁺ recognition mechanism of chemosensor 76.

Ravikanth et al. recently developed a BODIPY-based receptor (76; Fig. 40B) with a benzimidazole moiety at the 3-position for the recognition of Hg²⁺ ions in CH₃CN–PBS (7 : 3; v/v, pH 7.4) solution with the efficiency of the lower detection limit of 0.77 μM.⁶⁷ This system was also used for in vitro detection of Hg²⁺ ions with little interference of Cu²⁺ ions. Moreover, the receptor 76 exhibited blue shifts in absorption spectra from 576 nm to 529 nm with an isosbestic point at 545 nm and allowed a ratiometric recognition of Hg²⁺. Similar blue shift from 603 nm to 582 nm was also observed in emission spectra with an increase in quantum yield (Φ) from 0.42 to 0.58 owing to the interruption of photo-induced electron transfer (PET) process in presence of Hg²⁺. The observed association constant calculated from the emission titration was 6.18 × 10⁶ M⁻¹, while binding stoichiometry, i.e. receptor to Hg²⁺, was 2 : 1. The receptor could be useful for the detection of Hg²⁺ in the human breast adenocarcinoma cell line MDA-MB-231 for in vivo imaging measurements.

Misra and co-workers developed used aggregation-induced emission enhancement (AIEE) method for the detection of Hg²⁺ ions.⁶⁸ They synthesized bathophenanthroline (BA; 77; Fig. 41A)-based aggregated microstructures of various morphologies using re-precipitation method and characterized by optical, powder X-ray diffraction and scanning electron microscopy. The aqueous dispersion of 77 microstructures showed AIEE at 384 nm (λ_ex = 270 nm) compared to THF solution of BA. This luminescent property of aggregated BA hydrosol was used for the detection of metal ions and among various metal ions screened, and its AIEE was quenched only in presence of Hg²⁺ ion. The aggregated microstructure can detect even trace amount (10⁻⁷ M) of Hg²⁺ in aqueous medium. This strong fluorescence quenching of aggregated BA in the presence of Hg²⁺ ions was supposed to be due to the heavy atom induced perturbation by the complexed Hg²⁺ ions on the excited states of the 77.

Fig. 41 (A) Structure of 77 and analogues aggregated microstructure. Reprinted with permission from ref. 68. Copyright 2014 Royal Society of Chemistry. (B) Structures of Biginelli-based molecules 78, 79 and 80, for the recognition of Hg²⁺ ions.

Singh et al. recently developed Biginelli-based molecules (78–80; Fig. 41B) with the dimension of nanometre size for the recognition of Hg²⁺ in aqueous tris buffer medium at pH 7.4 (1 mM).⁶⁹ Only receptor 79 showed selectivity with Hg²⁺ while 80 showed interference for Cu²⁺; however, 78 could not show any selectivity for the metal ions under the experimental condition. The organic nanoparticles (ONPs) 79 (size 28 nm), showed ratiometric property with an increase in absorption band at 345 nm and a decrease in band at 386 nm with an isosbestic point 369 nm in presence of Hg²⁺ with the detection limit as low as 1 nM. A continuous decrease in emission spectra with a blue shift from 456 nm to 440 nm was also observed. These changes could be due to the coordination of Hg²⁺ ions with S and N centres (C=N group), leading to the new LMCT band. Addition of Cl⁻ ions into 79·Hg²⁺ restored original absorption spectra but showed completely different cyclic voltammogram and differential pulse voltammetry. Thus, ONPs were considered as secondary sensors for Cl⁻ ions rather than the reversible sensor for Hg²⁺ ions.

Kuwar and his group reported easily synthesizable, cost-effective, noncyclic naphthalene-based highly selective fluorescent chemosensor (81; Fig. 42).⁷⁰ This receptor showed a selective chromogenic and fluorescent turn-off response towards Hg²⁺ in an ensemble of several other metal ions in DMSO–H₂O (9 : 1) system. Analysis of the experimental results suggests a binding stoichiometry of 1 : 1 with a lower detection limit of micromolar level. This composite reagent was found to be cell membrane permeable and could be used as an imaging reagent for detection of the cellular uptake of Hg²⁺ by live HeLa cells, as well as the detection of Hg²⁺ present in water in environmental samples. Moreover, the DFT calculations were performed to complement the experimental evidences.

Fig. 42 The chemical structure of the chemosensor 81.

Recognition of Hg²⁺ using immobilized molecular probes

One of the aims of the modern chemists is to develop new materials with the immobilization of the chemosensors on the solid supports as this offers the opportunity of recognition of the target analyte, as well as the obvious ease in separating the analyte from solvent or mobile phase. Use of such probes, confined on the surface of a solid material or inside of a porous structure, obviously gets preference over using solution of the same probes as it offers the possibility of in-field purification or scavenging along with recognition/detection. In this regard, use of various polymeric backbones, glass particles, gold nanoparticles and different nanostructures with proper design have gained significance for ‘in-field’ use for sensing detection purposes.⁷¹ For real application opportunities, such molecular receptors should ideally be linked to the surface of nanostructured particles (NPs), to the thin films of membranes or to a porous polymeric substance. Such materials generally have surface-enhanced detection, which allows the sensing of an analyte that is present at a very low concentration. Significance of such hybrid systems are even more important when binding of the analyte leads to binding-induced changes in output signal that offer the possibility of real-time monitoring of the binding and possible scavenging process. Various methods that could be monitored for probing the binding process include measurable changes in electronic/fluorescence spectral properties, visually detectable colour/fluorescence changes, redox potentials and electro-generated chemiluminescence. Appropriate hybrid material and methodology could actually help in avoiding use of expensive and complex analytical instrumentation with long acquisition times and the use of trained personnel. To date, there are only a few examples available in the literature that address such an issue for the detecting and scavenging of Hg²⁺ in aqueous solution. Therefore, development of such a cost-effective alternate hybrid material with simple user-friendly operating process is almost essential considering the surge in enhancement in the artificial activities that lead to the increase in possibility of Hg²⁺ pollution in potable water, and thus developing simple analysis technique for in field application.⁷²

Solid-supported receptors for the detection of Hg²⁺

Mesoporous silica with immobilised tren-derivative of rhodamine B could be used for the detection of Hg²⁺ (82; Fig. 43).⁷³ Mesoporous silica surface was functionalized with the spirolactam form of the rhodamine B. Rhodamine B derivatives in their spirolactam form do not show any absorption or emission band beyond 400 nm, and thus such functionalized silica particles are expected to be either colourless or to possess a pale yellow colour. However, binding of Hg²⁺ to such silica–rhodamine hybrid is expected to convert the spirolactam ring to the acyclic xanthene form with simultaneous change in the colour of the modified silica particles. Reversibility in binding to Hg²⁺ could be demonstrated after treatment with TBA⁺ OH⁻ solution, and thus these modified silica receptors could be reused for several cycles.

Fig. 43 Rhodamine derivative immobilised on mesoporous silica surface, for the reversible recognition of Hg²⁺.

Du, Chen and their co-workers could develop an analogous silica surfaces, in which rhodamine 6G was grafted on silica surfaces for use as a solid supported sensor (Fig. 44) for Hg²⁺ ion.⁷⁴ For this reagent, the linear concentration range for Hg²⁺ was evaluated as 0.4–8.0 × 10⁻⁷ M with a lower detection limit (S/N = 3) of 2.59 × 10⁻⁹ M. In order to achieve the ratiometric fluorescence response following an energy transfer mechanism, CdTe quantum dots (QDs) were used in combination with these modified silica particles. QDs were used to provide the fluorescence background in the ratiometric fluorescence approach and were immobilized in silica nanoparticles to compose QDs@SiO2–Rh6G nanoparticles as shown in Fig. 44. These hybrid particles displayed well-resolved dual fluorescence emission with the Rh6G derivative at 545 nm, and the CdTe-QDs at 625 nm, used successfully in determining Hg²⁺ in water samples having [Hg²⁺] of 1.7 × 10⁻⁷ M.

Fig. 44 Rhodamine 6G (83) grafted on the silica surface in the hybrid receptor for the recognition of Hg²⁺. Schematic representation for the methodology adopted developing QDs@SiO₂–Rh6G.

A slightly different methodology was adopted for achieving the FRET response on recognition and binding to Hg²⁺ ion in aqueous medium. The close proximity of the donor and acceptor allowed the FRET process to be operational (Fig. 45).⁷⁵ Spirolactam form of the rhodamine derivative was covalently linked with the silica surface covering the Cd–Te QDs/silica core. In absence of Hg²⁺, the nanosurface neither showed any absorbance nor exhibited any fluorescence beyond 400 nm. On binding to Hg²⁺, FRET process was operational and a strong luminescence, having maxima at 535 nm and a strong pinkish red colouration, were obtained as output signals for probing the Hg²⁺ recognition process. This hybrid sensor was selective towards Hg²⁺ with the lower detection limit of 260 nM and was effective over a wide pH range. It was argued that such strategy could be used to construct QDs-based ratiometric assays for other ions, which quench the emission of QDs.

Fig. 45 Strategy used for FRET-based Hg²⁺ sensor on QDs.

Recently, carbon nanodots (CDs) functionalised with bis(dithiocarbamato) Cu(II)-complex 85 (Fig. 46) were introduced as a fluorescence ‘turn on’ sensor for Hg²⁺ with a lowest concentration detection limit of 4 ppb.⁷⁶ Here, initial Cu²⁺-complex was non-fluorescence owing to the combined effect of energy transfer and electron transfer mechanism. The higher affinity of the Hg²⁺ ions as compared to Cu²⁺ towards the receptor helped to displace Cu²⁺ ions readily from the surface of CDs led to a more stable sulphur-chelated Hg²⁺-complex with significantly enhanced emission intensity at ∼450 nm. Moreover, it was useful for the detection of Hg²⁺ ions on commercial cellulose acetate inkjet paper under a UV lamp of wavelength 365 nm. However it showed slight interferences with Ag⁺ and with AuCl₄⁻.

Fig. 46 Schematic representation of ‘turn on’ response of carbon nanodots 85 by displacement of Cu²⁺ ions by Hg²⁺ ions. Reprinted with permission from ref. 76. Copyright 2014 American Chemical Society.

Poly[(p-phenyleneethynylene)-alt-(thienyleneethynylene)]-conjugated polymer back bone (86) showed absorption at 441 nm and emission at 487 nm, which had a substantial spectral overlap with the absorption spectral band of the Hg²⁺-bound xanthene form of rhodamine. This initiated an effective FRET between the polymer as the donor and the Hg²⁺-bound rhodamine fragment as the acceptor unit (Fig. 47) and a new emission band appeared at 575 nm along with an isosbestic point at 552 nm.⁷⁷ However, use of THF as the solvent has actually restricted the application potential of this receptor.

Fig. 47 Structure of the polymer back bone in conjugation with rhodamine for the recognition of Hg²⁺ ions.

Kaewtong and his co-workers developed a series of new amino ethyl rhodamine B derivatives with varying spacer between two amine functionalities, which showed specificity in binding to Hg²⁺ with a binding constant of 83 000 M⁻¹ for a 1 : 1 binding stoichiometry in acetonitrile medium (Fig. 48).⁷⁸ These derivatives also showed a weak affinity towards Cu²⁺; however, affinities for Hg²⁺ were at least 300 times higher than Cu²⁺ for receptors 87–90. Note that a polymeric thin film can be obtained by doping poly(methyl methacrylate) or PMMA with chemosensor 89. This non-fluorescent thin polymer film showed emission on spraying Hg²⁺ on top of it. The reversibility in binding process was demonstrated by treatment with NaOH solution, which restored the original colour and intensity of the doped polymer film. However, the limitation of using acetonitrile as the only solvent has limited its use for most practical applications.

Fig. 48 Structure of the receptors (87–90) doped onto polymer sheets.

Another silica-supported solid sensor 91 was prepared in the reactions of aminopropyltriethoxysilane monolayer-coated mesoporous silica nanoparticle (MCM-41 SiNPs) and 4-(4-isothiocyanatophenylazo)-N,N-dimethyl aniline (Fig. 49).⁷⁹ Absorption titration with Hg²⁺, in the suspension of 91 showed purple colouration and decrease of the band at 416 nm, while of new band appeared at 540 nm with consequent ratiometric behaviour. The minimum detection limit obtained was as low as 2 ppm with a 1 : 1 binding stoichiometry and an association constant of 20 038 M⁻¹ in THF medium. The functionalised nanoparticles 91 could be regenerated by addition of polar protic solvents like water and could be used for several cycles. However, use of this supported receptor was also restricted as it could only be used in THF medium.

Fig. 49 Modified mesoporous silica for chromogenic recognition of Hg²⁺ [inset: (a) change in colour of 91 in presence of different metal ions, (b) Cycles of Hg²⁺ absorption after washing with water]. Reprinted with permission from ref. 79. Copyright 2013 Royal Society of Chemistry.

Analogous receptor 92 with rhodamine moiety immobilized on silica surface was used to make a dual probe for the recognition of Hg²⁺ from the aqueous medium (Fig. 50).⁸⁰ The absorption and emission experiments were performed in the water suspension, in which the pale yellow colour of the modified silica particles turned red in colour in presence of Hg²⁺ with concomitant enhancement of the emission signal at ∼550 nm. The red solid obtained after detection of Hg²⁺ could be regenerated into its original form (Fig. 50) by the treatment of TBAOH and it could be recycled for at least three times. The adsorption ability of 92 for Hg²⁺ in neutral water was also estimated by atomic absorption spectroscopy, which revealed an efficiency of about 72%. However, it showed a weak interference from Cu²⁺ ions.

Fig. 50 Rhodamine-based solid support for the recognition of Hg²⁺ [inset (a) colour changes for 92 solid without, (b) with metal binding after isolation from the aqueous suspension; and (c) upon successive immersion in 1 M TBA⁺OH⁻ aqueous solution]. Reprinted with permission from ref. 80. Copyright 2012 Royal Society of Chemistry.

Fu et al. have recently developed a rhodamine-based sensor 93, supported on cellulose surface for the recognition of Hg²⁺ in aqueous medium. Abundance, bio-degradability and cost-effectiveness (Fig. 51) are the key advantages of using cellulose as the support material in an attempt to develop an efficient metal ion scavenger.⁸¹ Rhodamine derivative was covalently linked to cellulose surfaces through appropriate functionalization in order to avoid leaching of the sensors in aqueous environment. After proper characterisation, the colourless cellulose paper was immersed into the tris–HNO₃ buffer (pH 7.24) solution containing Hg²⁺, while it turned red and a new characteristic absorption band centred at 553 nm was observed. This was found to enhance with increase in [Hg²⁺] within 2 min. Studies in emission spectroscopy revealed a new switch on emission band at 574 nm with a lower detection limit for Hg²⁺, 5.0 × 10⁻⁵ M. More importantly, it showed reversibility in binding to Hg²⁺ on treatment with 1.0 × 10⁻³ M solution of Na₂S with 85% recovery of its original emission pattern.

Fig. 51 Rhodamine derivative on modified cellulose filter paper [inset: optical photograph of cellulose paper in presence of different metal ions]. Reprinted with permission from ref. 81. Copyright 2013 Royal Society of Chemistry.

Zhu et al. have used salen for its well-known ability to bind to certain metal ions, and thus to achieve chelation enhancement fluorescence on binding to those transition metals (Fig. 52).⁸² For the receptor 94, highly emissive perylene was covalently attached to salen building block in order to develop a fluorescent as well as a colorimetric sensor for the recognition of Hg²⁺. This reagent showed selectivity towards Hg²⁺ over Na⁺, K⁺, Ca²⁺, Ag⁺, Ni²⁺, Cd²⁺, Pb²⁺, Cr³⁺ Al³⁺, Fe³⁺, Co²⁺ and Zn²⁺.

Fig. 52 Polymer-based system for the recognition of Hg²⁺ through coordination induced ICT ‘ON–OFF’ process. Reprinted with permission from ref. 82. Copyright 2012 Royal Society of Chemistry.

The parent polymer used in 94 showed weak ICT-based emission at 635 nm (λ_ext of 440 nm), involving salen moiety as donor and perylene as an acceptor moiety. On binding to Hg²⁺, a 26-fold enhancement in the emission signals with 85 nm blue shift in emission maxima was observed, and this allowed a lower detection limit of 7.28 × 10⁻⁷ M for Hg²⁺.

Lee, Jung and their co-workers have demonstrated that an aminonaphthalimide-functionalised Fe₃O₄ nanoparticle, within the core of SiO₂, could detect and be able to separate Hg²⁺ from drinking water (Fig. 53).⁸³ The naphthalimide-based emission became quenched upon addition of Hg²⁺, while the original emission of the reagent was restored on addition of EDTA (0.01 N). The Job's plot confirmed a 1 : 1 binding stoichiometry, while the binding affinity was reported to be 1.05 × 10⁵ M⁻¹. Moreover, 95 could detect Hg²⁺ as low as 1.02 ppb. However, the emission of 95 was quenched below pH 4 due to protonation on the nitrogen atoms, and thus this reagent could be useful for detection of Hg²⁺ within the pH range 6–11.

Fig. 53 Structure of the functionalised nanoparticle for the detection of Hg²⁺ [inset: photograph of a magnet attracting 95 in water]. Reprinted with permission from ref. 83. Copyright 2010 Royal Society of Chemistry.

Das et al. have reported a self-indicating scavenger for Hg²⁺ ion from its aqueous solution using Ca-salt of rhodamine–alginate bio-polymeric gel bead (96, Fig. 54).⁸⁴ This reagent also showed some affinity towards Cr(III). However, through judicious use of appropriate masking agent like I⁻, it was possible to detect Cr(III) quantitatively even in presence of Hg²⁺. Thus, this reagent could be used for detection of total amount of Hg²⁺ + Cr³⁺ present in an ensemble of several other metal ions, as well as the individual concentration of Cr³⁺ and Hg²⁺, in aqueous solution. Note that alginate is a natural polymer and easily accessible. Such hybrid beads turned pink-red on binding to Hg²⁺ and/or Cr³⁺, and thus could be used for self-indicating Hg²⁺/Cr³⁺-sponge. Furthermore, studies reveal that such beads could be reused and were useful as a stationary phase in a column for removal of these two ions. The unique technique demonstrated that such hybrid material derived from rhodamine–alginate gel could be used as a stationary phase for reducing the [Hg²⁺] below the toxic level (2 ppb).

Fig. 54 Bio-polymeric gel bead of rhodamine–alginate conjugate was used for the detection and for the removal of Hg²⁺ from its aqueous solution.⁸⁴

Huang et al. developed a novel reversible cellulose-based colorimetric Hg²⁺ sensor by immobilizing monolayer of a ruthenium dye 97 onto TiO₂ ultrathin gel film pre-coated cellulose nanofibres (Fig. 55).⁸⁵ The resultant sensor material exhibited extraordinary selectivity and sensitivity with associated colour change from purple to orange in the presence of Hg²⁺ in aqueous solution. The reported detection limit was ∼10 ppb for the naked eye. Reversibility of the binding process was established in presence of KI solution, and this was efficient for trapping Hg²⁺ ions in aqueous media.

Fig. 55 Hg²⁺ ion recognition using TiO₂-97 multilayer modified natural cellulose substance.⁸⁵

Cheng and co-workers utilized click reaction to develop a highly selective and sensitive polymer-based fluorescence sensor 98 having triazole unit (Fig. 56A) for Hg²⁺ detection.⁸⁶ In MeOH–H₂O (1 : 1, v/v) medium, 98 showed emission at 425 nm with a shoulder at ∼450 nm on excitation at 381 nm, and this emission was appreciably (86%) quenched in presence of Hg²⁺. This could have happened due to either of two or a combination of two influences; i.e., binding to Hg²⁺ had an adverse influence on the ICT process and the efficient spin–orbit coupling process involving the Hg²⁺ ion. Visually detectable fluorescence of the polymer disappeared on binding to Hg²⁺. The lowest limit for Hg²⁺ detection of 98 was 4.69 × 10⁻⁷ mol L⁻¹. An interference of Ag⁺ was also reported for this reagent.

Fig. 56 (A) Molecular structure of the reagent 98 and (B) Hg²⁺ detection mechanism of silica FRET based sensor 99.

Zeng and Wu et al. developed a multilayered silica film (99; Fig. 56B) on a quartz plate for the FRET-based ratiometric detection of Hg²⁺ in aqueous medium.⁸⁷ They consecutively deposited silica functionalized donor (a nitrobenzoxadiazolyl derivative, NBD) layer, spacer layer, and finally the acceptor (rhodamine) layer on the outside for the binding of metal ions. In aqueous medium, only in presence of Hg²⁺ among various metal ions it shows absorption at 560 nm and emission at 580 nm due to the opening of the spirocyclic ring of the rhodamine moiety. However, upon excitation at 430 nm, FRET from the NBD unit to the ring-opened rhodamine moiety happened and the system showed ratiometric behaviours. Furthermore, this device could able to detect even 1 μM conc. of Hg²⁺ ions in aqueous medium.

Ding and co-workers developed a novel polyaniline (PANI)-based sensor (100; Fig. 57) for the colorimetric detection of Hg²⁺ ions in aqueous medium.⁸⁸ These hierarchical structured nanofibrous sensing membranes composed of electrospun nanofibres and ultrafine nanowires demonstrate enhanced homogeneity, interconnectivity and porosity, which greatly boosts the colorimetric sensing properties. The sensor shows decrease in reflectance intensity at 440 and 645 nm with change in colour from white to yellow/green, green and blue upon exposure to aqueous solution of Hg²⁺. The leucoemeraldine-based PANI probe had specific interaction with Hg²⁺ ions and caused the change in colour, and it could detect Hg²⁺ ion in aqueous medium as low as 5 nM with visually detectable colour change. The sensor showed a good reversibility, which was exerted in several cycles. Furthermore, authors also demonstrated that this reagent could be used for qualitative detection of the variation in [Hg²⁺] based on the solution colour gradient.

Fig. 57 Structure of the chemosensor 100 and its Hg²⁺ binding mechanism.

Heng and his co-workers reported a fluorescence-based silole-infiltrated photonic crystal hexaphenylsilole (HPS)-infiltrated photonic crystal (PC) film (101; Fig. 58) for effective and reversible detection of Fe³⁺ and Hg²⁺ ions.⁸⁹ PCs show amplified fluorescence due to the slow photon effect of PC and electron transfer of HPS molecules and metal ions. The original fluorescence was amplified by the slow photon effect of PC and was quenched significantly due to electron transfer between HPS and Fe³⁺, Hg²⁺ and Ag⁺ ions having higher electrode potential (ions have higher standard electrode potentials: Fe³⁺, 0.77 V; Hg²⁺, 0.85 V; Ag⁺, 0.8 V). However, the emission quenching efficiency was maximum for Hg²⁺ and Fe³⁺ (70–65%) and was least for Ag⁺ (∼5.6%), which could be attributed to the larger ionic diameter for the Ag⁺. The amplified fluorescence could help in enhancing the sensitivity with a detection limit of 5 nM for both these ions (Fig. 59).

Fig. 58 Proposed sensing mechanism for the detection of metal ions (Hg²⁺/Fe³⁺) by HPS-PC film sensor (101).

Fig. 59 The pictorial representation of the Hg(II) sensing nanocomposite material (102) constructed with silica coated β-NaYF₄:Yb³⁺/Er³⁺ nanorods and rhodamine derivative.

Since the luminescence of probe may also be quenched by other metal ions and emission quenchers in a complicated background, these ‘on–off’ sensors suffer from limited sensitivity towards specific analyte.⁹⁰ Moreover, these probes usually need the photoexcitation in UV and blue regions, where some other chromophores may also be excited, leading to the defect of background light interference. It seems that the combination of ‘off–on’ sensor and excitation source based on near-infrared to visible upconversion material can meet the above requirement.⁹¹ As for the excitation source, the upconversion material can transfer infrared radiation to visible photoexcitation; thus, probes can be efficiently excited with the chromophores unexcited. For this purpose, NaYF₄ lattice has been widely recognized as a promising host for upconversion materials.⁹²

In this regard, Wang and his co-workers developed a turn-on Hg(II) sensor system (102; Fig. 59) excited by near-infrared to visible upconversion nanorods.⁹³ For this purpose, they had successfully assembled a Hg(II)-sensing system (102) with the upconversion nanorods of β-NaYF₄:Yb³⁺/Er³⁺ as the excitation source and a rhodamine derivative as the signalling probe. The morphological aspect and photophysical property of this composite material have been well characterized by electron microscopy, XRD analysis, IR spectra, TGA/DTG analysis, and the energy transfer process between the excitation source and the probe was also investigated by excitation/emission spectra and excited state lifetime analysis. It is found that β-NaYF₄:Yb³⁺/Er³⁺@Rb (102) possesses a rod-shaped morphology with silica shell as slim as ∼10 nm. The rhodamine-based probe is covalently grafted onto the surface of β-NaYF₄:Yb³⁺/Er³⁺ nanorods and the doping content is 9.6%. The upconversion emission from β-NaYF₄:Yb³⁺/Er³⁺ nanorods can effectively excite the probe. Then, the sensing behaviour towards Hg(II) was evaluated by emission spectra and the Stern–Volmer plot. A good linear response in the range of 0–12 μM is obtained. Moreover, β-NaYF₄:Yb³⁺/Er³⁺@Rb (102) shows a good selectivity towards Hg(II), which makes itself a promising specific sensing system.

Xu and co-workers have successfully constructed a nanocomposite (103; Fig. 60) for specific recognition of Hg²⁺.⁹⁴ The nanocomposite with rod-shaped morphology and diameter of ∼160 nm was appropriately characterized using electron microscopy, XRD, IR/UV-vis/emission spectroscopy and thermogravimetry. This nanocomposite was covered with a uniform silica shell (thickness ∼15 nm) to improve the dispersibility in aqueous solutions. Rhodamine-based receptor was then grafted onto the silica shell with doping content of 4.01% through silylation reaction. The Forster radius calculation and excited state lifetime analysis suggested an efficient energy transfer between the excitation core and the probe. This constructed sensing system owned a linear response towards increasing [Hg(II)] with a “turn-on” response. This composite showed high specificity towards Hg(II) as well as high photostability. However, possible use of such nanocomposite as an imaging reagent was not described.

Fig. 60 Representation of the Hg(II) sensing nanocomposite system (103) constructed with β-GPTS modified NaYF₄:Yb³⁺/Er³⁺ nanorods and rhodamine derivative.

Cui et al. reported a turn on sensor for the detection of Hg²⁺ using the abovementioned energy transfer mechanism involving similar type of β-NaYF₄:Yb³⁺/Er³⁺–rhodamine composite nanorods (103; Fig. 60) as a sensing probe for Hg²⁺.⁹⁵ Based on the results of the upconversion, as well as the time-resolved emission studies, FRET-based energy transfer from the Hg²⁺-bound rhodamine moiety to the phosphor material was established and this emission response was utilized for Hg²⁺ ion recognition. However, interference from other metal ions like Fe²⁺, Cu²⁺, Cd²⁺, Pb²⁺ and Ag⁺ was also reported.

Arunbabu and co-workers developed a novel polymer based photonic crystal hydrogel sensor (UPCCA) for the selective detection of toxic Hg²⁺ ions in aqueous medium (105; Fig. 61B).⁹⁶ This colloidal polymeric photonic crystal could diffract visible light at 764 nm in aqueous medium, and the diffraction wavelength shifted owing to the volume phase transitions experienced by the polymer hydrogel upon exposure to the different mercury ion concentrations. UPCCA consisted of urease enzyme, which could hydrolyse the urea substrates and produce the HCO₃⁻ and NH₄⁺ ions inside the hydrogel. These ions decreased the electrostatic repulsion between carboxylates and the polyacrylamide backbone and led to the shrinkage of hydrogel. Hg²⁺ ion inhibited the urease–urea hydrolysis reaction, and thus suppressed the ion production and did not allow the hydrogel to shrink, as it was the case in absence of Hg²⁺; moreover, it simultaneously affected the diffraction wavelength of the incident light on these colloidal particles.

Fig. 61 (A) The chemical structure of the rhodamine derivative 104. (B) Polymer-based photonic crystal hydrogel sensor 105.

Detection of toxic Hg²⁺ ions by highly photo-active chemodosimeters

To avoid the problem of high solvation energy of Hg²⁺ in aqueous medium, and thus to achieve the detection process in polar solvent like water as well as in physiological condition, chemodosimetric approach as a detection methodology and diagnostic tool has gained importance more recently. In this approach, probe molecule reacts with the desired analyte and undergoes chemical transformation with associated change(s) in output signal, which allows the quantitative estimation of the target analyte. These chemodosimeters are generally categorised into two types in terms of the changes in their optical output signals: firstly, optical chemodosimeters that allow detection/quantification of the analyte either through ‘on–off’ or through ‘off–on’ changes in their optical (fluorescence or/and absorbance) output signals owing to the specific chemical reaction on the course of analyte recognition. Secondly, ratiometric chemodosimeter that shows ratiometric changes in absorption or/and emission spectra with a certain isosbestic or isoemissive point on reaction with the desired analyte.⁹⁷ Again, in some cases, analytes react directly with the probes, while in some cases analytes catalysed the chemical transformation with consequent change in the optical properties.^3b These approaches often get preference over normal probes in terms of selectivity and sensitivity, more specifically in aqueous medium.

Chemodosimeters for the detection of Hg²⁺ ions

According to hard–soft acid–base theory, Hg²⁺ is soft acid; consequently, it has very high affinity towards the soft base sulphur. This high thiophilic character of Hg²⁺ has been utilized for its recognition. Chemodosimeter is one class of chemosensor, which recognizes analyte after an irreversible mechanism.

Tae et al. developed a rhodamine-6G derivative 106, which works as a highly selective and sensitive chemodosimeter for Hg²⁺ in aqueous solution (Fig. 62A).⁹⁸ Hg²⁺-promoted an irreversible oxadiazole-forming reaction for 83 with visually detectable changes in fluorescence and colour. Thus, monitoring either of these spectral changes one could probe the Hg²⁺ ion recognition process at room temperature in a 1 : 1 stoichiometric manner to the amount of Hg²⁺ present. Shin et al. could further utilize this system for demonstrating a real-time method for monitoring the concentration of mercury ions in living cells, particularly vertebrate organisms (Fig. 62B).⁹⁹

Fig. 62 (A) Schematic representation of Hg²⁺-promoted oxadiazole-forming reaction of the reagent 106 and (B) images of zebrafish organs treated with 5 nM HgCl₂ and 10 μM 106. Reprinted with permission from ref. 99. Copyright 2006 American Chemical Society.

Bharadwaj, Kim and co-workers developed a cryptand–rhodamine conjugate (107) for the detection of Hg²⁺.¹⁰⁰ The geometric arrangement of aliphatic N-atoms in the cryptand core in 107 offered the appropriate binding sites for Hg²⁺ (Fig. 63). Selective binding of Hg²⁺ to 107 followed a 1 : 3 stoichiometry in EtOH–H₂O medium (2 : 5, v/v). Composite association constant of ∼7.0 × 10¹¹ M⁻³ was reported. Control experiment with HEK 293 cells incubated with only 107 displayed a very weak fluorescence image; however, strong cellular fluorescence was observed when similar cells pre-exposed to Hg²⁺ cells were incubated with 107.

Fig. 63 Molecular structures of reagents 107, 108 and 109.

Yoon and co-workers have introduced a rhodamine 6G thiolactone derivative 108 (Fig. 63) as a selective and colorimetric sensor for Hg²⁺ at pH 7.4.¹⁰¹ An ‘off–on’ type fluorescence and colorimetric responses were observed in the presence of Hg²⁺ in CH₃CN–HEPES buffer (1 : 99, v/v, pH 7.4) medium. X-Ray structure of Hg²⁺·[108]₂ confirmed the 1 : 2 binding stoichiometry. Importantly, reagent 108 could detect Hg²⁺ in the nanomolar range of concentration, and it could be used for in vivo imaging of C. elegans for detection of Hg²⁺ ion uptake. Furthermore, Yoon and Shin's group together designed a unique selenolactone based probe 109 (Fig. 63) for the detection of inorganic mercury and methylmercury species through anticipated fluorescence and UV-vis spectral change.¹⁰² Spectral studies also confirmed the 1 : 1 stoichiometry between 109 and Hg²⁺-species. This reagent was further used for the detection of inorganic mercury, as well as the potent neurotoxin, i.e., methylmercury, in cells and zebrafish.

The reactivity of the disulphide linkage towards Hg²⁺ was also utilized for Hg²⁺ ion recognition. A new rhodamine-based chemodosimeter (110) was utilised for this purpose (Fig. 64).¹⁰³ The receptor 110 exhibited selectivity only for Hg²⁺ with generation of the absorption band at 560 nm and emission band at 580 nm in ethanol–water medium (80 : 20, v/v) due to the generation of the acyclic xanthene form. Other alkali, alkaline earth metals and transition metals remained inactive toward ring-opening under the experimental condition.

Fig. 64 Reaction of disulphide linkage with Hg²⁺ ions led to the formation of acyclic xanthenes form.

Electron-rich alkynes are known to react with Hg²⁺ to undergo a further hydrolysis reaction to yield the aldehyde functionality. Reaction of Hg²⁺ with sulphur and alkyne functionality led to the formation of thiazole ring with a mercurated exocyclic double bond (Fig. 65).¹⁰⁴ However, the exocyclic alkene bond underwent a facile hydrolysis reaction to yield the corresponding formyl derivative with significant red emission in the visible region (∼560 nm). The mechanism of co-operative interaction was explained on the basis of control experiments. The alkyne derivative without S-atom and vice versa could not produce any change in emission, which further corroborated the proposed mechanism for the recognition process as well as the fluorescence on response.

Fig. 65 Reaction pathway for the formation of formyl thiazole with lactam ring opening.

Schiff's bases are also known to be prone towards hydrolysis and this reaction becomes more effective in presence of Hg²⁺. This property was utilized for designing the chemodosimeter 112 for specific recognition and quantitative estimation of Hg²⁺, where it basically acts as a catalyst to introduce this hydrolysis reaction (Fig. 66).¹⁰⁵ The reported lower detection limit for Hg²⁺ ions was as low as ppb level in natural water. Coordination of Hg²⁺ centre to 112 led to the opening of the lactam ring and the generation of the highly emissive and strongly absorbing xanthene form, which underwent further hydrolysis to generate carboxy derivative as the final product with 379-fold enhancement in emission intensity at ∼580 nm. Moreover, this methodology nullified any interference of sulphide containing bio-analytes such as cysteine/homocystine and glutathione, especially for live cells imaging.

Fig. 66 Hydrolysis protocol of the chemodosimeter 112 in presence of Hg²⁺ led to the formation of rhodamine B.

Reaction of Hg²⁺ with alkynes first leads to the mercuration reaction, which on hydrolysis, i.e. on demercuration, produces the corresponding ketone (Fig. 67A).¹⁰⁶ This methodology has been introduced for the development of a chemodosimeter 113 for Hg²⁺ with ‘turn on’ response showing 219-fold enhancement in emission intensity with maxima at 523 nm.¹⁰⁶ More importantly, probe 113 was used for the detection of Hg²⁺ ions in Solomon fish-tissue after dissolving in Me₄NOH in presence of strong oxidant NCS (N-succinimide) at the pH 7.0. The strong fluorescence signal suggested that 113 could be used to monitor mercury concentrations in fish and in other tissues as 95% of mercury species exist as MeHg⁺ in fish. The receptor 113 could detect Hg²⁺ as low as 8 ppb level (S/N = 3). Vinylic ether also has affinity for Hg²⁺ ions and eventually leads to the formation of acetaldehyde and the corresponding phenolate (Fig. 67). Thus, non-fluorescent receptor moiety 114 showed fluorescence in presence of Hg²⁺ with ‘turn on’ signal at 525 nm, and the observed detection limit was lower than 1 ppb (Fig. 67B).¹⁰⁷ Importantly, chemodosimetric reagent 114 could detect Hg²⁺ in river water and from dental samples.

Fig. 67 Detection of Hg²⁺ based on the of oxymercuration and demercuration reaction of electron-rich π-bonds and the proposed mechanistic pathway for the elimination reaction.

Basak et al. have synthesized a 3-amino derivative (115; Fig. 68A), which took part in an irreversible ring opening reaction while specifically interacting with Hg²⁺.¹⁰⁸ This led to an in situ formation of 1,3,5-oxazole ring with ‘turn on’ enhancement of fluorescence in the water–ACN (60/40; v/v) medium. Chemodosimeter 115 showed 26-fold enhancement upon addition of one equivalent Hg²⁺ with shift in emission maxima from 579 nm to 583 nm within 3 minutes under the experimental condition. The spirolactam ring structure for this chemodosimeter 115 was found to be maintained within the pH range 4–10. Confocal microscopic studies revealed that this reagent was effective for recognition of Hg²⁺ in live HeLa, HEK293T, MN9D, and RN46A cells and also the in vivo studies in zebrafish.

Fig. 68 (A) Hg²⁺-mediated oxazole formation. (B) Hg²⁺ ions-promoted hydrolysis of vinylic ethers and excited state intramolecular proton transfer leading to change in emission signals.

A set of new oxymercuration derivatives (116a–116c) were reported by Ahn and his co-workers (Fig. 68B).¹⁰⁹ These derivatives showed large shifts in emission maxima with ratiometric response. The absorption maximum of 116a was shifted from 295 nm to 330 nm with an isosbestic point at 318 nm in presence of HgCl₂, and the consequent colour was changed from blue to cyano. The corresponding emission signal was found to shift from 420 nm to 500 nm in PBS buffer medium (1% CH₃CN, pH 7.4). More interestingly, it was observed that 2-(benzthiazol) phenol and their derivatives in their tautomeric keto forms showed emission at longer wavelength compared to the corresponding phenol derivatives due to an excited state intramolecular proton transfer mechanism. It was also observed that the hydrolysis reaction with HgCl₂ was faster in presence of electron-withdrawing group at the para-position with respect to the vinyl ether bond. However, the lowest detection limit that could be achieved was 20 ppb, which is little higher than that required to qualify as an efficient reagent for detection of Hg²⁺ for safe drinking water.

Similarly, Hg²⁺ ion-promoted hydrolysis of vinyl ether 117 (Fig. 69) generated the corresponding hydroxyl derivative, which was followed by an intramolecular nucleophilic addition reaction for generation of the highly fluorescent coumarin moiety with an associated ‘turn on’ emission response. This ideology was exploited for designing the receptor 117 for sensing of Hg²⁺ in PBS buffer solution in DMSO–H₂O (5 : 95, v/v; pH 7.4).¹¹⁰ An increase in emission intensity at 475 nm on reaction of HgCl₂ with 117 was observed and formation of the coumarin derivative as the eventual product was even isolated and characterized.

Fig. 69 HgCl₂-induced hydrolysis of vinyl ether and cyclization reaction.

Propargyl amide containing chemodosimeter 118 was also found to be converted into corresponding oxazole derivative on reaction Hg²⁺ (Fig. 70).¹¹¹ The oxazole formation was associated with large bathochromic shifts in absorption band from 428 to 475 nm with an isosbestic point at 438 nm. Systematic increase in [Hg²⁺] showed gradual bleach in intensity at 428 nm, and an enhancement of the emission intensity at 475 nm, which allowed a ratiometric detection of Hg²⁺. This transformation was complete within 40 min, and the rate constant for this reaction was 3.9 × 10⁻² M⁻¹ s⁻¹. Emission spectra for 118 were recorded in absence and presence of Hg²⁺, which clearly revealed a ratiometric response with a shift in the emission band maxima from 469 to 492 nm (λ_ext = 428 nm). An increase in the quantum yield value from 0.05 to 0.15 and the lower detection limit of 2.7 μM were reported.

Fig. 70 Hg²⁺-mediated oxazole ring formation leading to change in photophysical property.

Cyclodextrins are biologically benign and have the ability to penetrate easily through the phospholipid and cholesterol containing cell membranes. A new probe 119 was designed from napthylthiourea, covalently linked to β-cyclodextrine for the recognition of Hg²⁺ in aqueous medium and its detection in live cells (Fig. 71A).¹¹² The chemodosimetric reagent 119 showed weak fluorescence at 380 nm due to PET process that was operational between sulphur as donor and naphthalene as acceptor units, while addition of Hg²⁺ led to significant emission enhancement without any interference of other metal ions. Detailed studies confirmed the desulphurisation mechanism, and the minimum detection limit for Hg²⁺ for this method was 3.7 × 10⁻⁷ M. Chemodosimeter 119 could also be used for the detection of Hg²⁺ ions in yeast cells.

Fig. 71 (A) Proposed mechanism of Hg²⁺ detection using receptor 119 [inset: (a) dark-field image of 119, (b) dark-field image of 119 in presence of Hg²⁺ under fluorescence microscopy]; reprinted with permission from ref. 112. Copyright 2011 Royal Society of Chemistry. (B) Hg²⁺-mediated deprotection of aldehyde led to bright change in emission [inset: confocal microscopic image of the tail of a 5 day-old zebrafish larva incubated with 10 μM of 120 for 20 min followed by incubation with 1 μM Hg²⁺ (20 min)]. Reprinted with permission from ref. 113. Copyright 2012 Royal Society of Chemistry.

Moreover, new fluorescent ‘turn-on’ probe 120 was successfully demonstrated for the detection of Hg²⁺, in which dithian protection of benzoxadiazole was removed upon addition of Hg²⁺. The internal charge transfer from N,N-dimethyl amino group to dithian was very low due to low electron-withdrawing capacity; thus, it showed low fluorescence (Fig. 71B).¹¹³ However, on addition of Hg²⁺ in the solution of 120 in aq. HEPES buffer : CH₃CN (4 : 1, v/v; pH 7.40) media a substantial increase (230-fold) in emission intensity at 585 nm (λ_ext of 455 nm) was observed, along with the increase in relative quantum yield by ∼35% due to a favoured ICT process. The band maxima in the absorption spectra was shifted from 448 nm to 458 nm with generation of new π–π*-based transition band at 316 nm upon reaction with Hg²⁺. An associated colour changing from pale yellow to bright yellow was observed. The virtual ‘turn-on’ emission response of 120 in presence of Hg²⁺ was utilised as an imaging reagent for the detection of Hg²⁺ in 5 days old zebrafish larvae using laser confocal microscopy.

Analogous strategy that was adopted for reagent 120 was also adopted for 121 for the selective recognition of Hg²⁺ in aq. PBS buffer (pH 7.4) (Fig. 72A).¹¹⁴ Strong thiophilicity of Hg²⁺ selectively induced the deprotection of the thioketal group of 121 and generated the acyl group, which consequently initiated an intramolecular charge transfer (ICT) process between the electron-donor methylamino group and the electron-acceptor acyl group. This resulted in an increase in the emission intensity for 121 at 503 nm (λ_ext = 380 nm) with increase in [Hg²⁺]. The absorption maxima of 121 also shifted from ∼295 to 355 nm in presence of Hg²⁺ ions due to the generation of pull–push ICT process. Fluorescence studies also allowed detection of the acute neurotoxin CH₃Hg⁺. Compound 121 was pH-insensitive and thus could be adopted for Hg²⁺ sensing in biotic environments. It could detect trace of Hg²⁺ present in live HeLa cells through two photon microscopy.

Fig. 72 Hg²⁺-mediated hydrolysis of the dithian moiety leading to change in photophysical property.

Li and co-workers synthesized colorimetric chemodosimeter 122a and 122b using similar dithioacetal binding moiety for the selective recognition of Hg²⁺ ions (Fig. 72B).¹¹⁵ On addition of Hg²⁺, the colour of the acetonitrile solution of 122a changed from yellow to red due to the formation of aldehyde compound on hydrolysis of dithioacetal moiety because of a favoured ICT process. As a result, the absorption spectra shifted from 420 nm to 455 nm at low [Hg²⁺], and this spectra further shifted from 455 to 515 nm at even higher [Hg²⁺]. Note that compound 122b showed similar colour change and spectral shifts in CH₃CN medium; however, it showed better sensitivity towards Hg²⁺, and it could recognize as low as 20 μm of Hg²⁺ through visually detectable colour change. Having two extra hydroxyl groups, compound 122b had better solubility and could detect Hg²⁺ colorimetrically in CH₃CN–H₂O (9/1 (v/v), 20 mM HEPES, pH 7.0) medium. Test strips were prepared by immersing filter paper into a CH₃CN solution of 122b (1 × 10⁻³ mol L⁻¹) and drying in air. These were used for detection of Hg²⁺ in aqueous medium using ‘dipstick’ method.

Cheng and co-workers developed a new series of ICT-based chemodosimeters (123, 124 and 125; Fig. 73) by attaching various electron-donating thiophenes groups to a triphenylamine backbone with a dithioacetal moiety as a binding site for the metal ions.¹¹⁶ Upon addition of Hg²⁺ to the THF solution of 123, the emission intensity at 440 nm was found to decrease and a new emission appeared at 525 nm due to the favoured ICT process after Hg²⁺ induced the hydrolysis of the dithioacetal unit and led to the generation of the more electronegative aldehyde moiety. Thus, compound 123 showed the ratiometric behaviour on reaction with Hg²⁺. Furthermore, the absorption maxima of 123 at 385 nm gradually disappeared, while a new absorption band centred at 422 nm was observed. This accounted for the change in solution colour from colourless to yellow. Similar to 123, dithioacetals 124 and 125 were also used for the detection of Hg²⁺ ions. Compared to 123 and 124, the higher conjugation in 125 results in a better ICT effect after reaction with Hg²⁺ ion, which led to the formation of the aldehyde moiety, and this was further confirmed by molecular simulation studies. Furthermore, the authors had synthesized a polymeric chemodosimeter P123 by introducing compound 123 to the backbone of conjugated polymers (P) and used that for the recognition of Hg²⁺. In THF medium, an increase in [Hg²⁺] caused a steady decrease in the emission band intensity for P123 at 462 nm, and a concomitant increase in band intensity of a new emission band at 564 nm was observed. Such spectral changes could be used for ratiometric detection of Hg²⁺, with the lowest detection limit of 0.1 μM; moreover, reagent 123 could be used successfully as an imaging reagent for the detection of Hg²⁺ in HeLa cells.

Fig. 73 Structure of the chemodosimeters 123, 124, 125 and P123.

Das and co-workers developed a dithiane derivative of BODIPY (126; Fig. 74) for the selective detection of Hg²⁺ in physiological condition.¹¹⁷ This dithiane reagent reacts specifically with Hg²⁺ to regenerate the parent BODIPY-aldehyde with consequential change in visually detectable optical responses, and this provides the possibility of using this reagent as a colorimetric probe or as a fluorescent biomarker/imaging reagent. Furthermore, non-covalent interactions could be utilized for formation of an inclusion complex with biologically benign β-cyclodextrin for enhancing its solubility in aqueous environment, and this included adduct could be used as a fluorescent marker and imaging reagent for Hg²⁺. Uptake of Hg²⁺ ions in live HeLa cells, which are exposed to a solution having Hg²⁺ ion concentration as low as 2 ppb, could also be detected by confocal laser microscopic studies.

Fig. 74 (A) Chemodosimetric recognition and detection of Hg²⁺ by the reagent 126 with possible inclusion complex formation with β-CD with confocal images of live HeLa cells, supplemented with 2 ppb of Hg(ClO₄)₂ in the growth media for 15 min at 37 °C and was followed by staining with 5.0 μM 126 for 1.0 h at 37 °C (λ_exc of 488 nm, λ_mon filter range of 494–502 nm). Reprinted with permission from ref. 117. Copyright 2013 Royal Society of Chemistry.

Like sulphur, high stability of HgSe complex could also be utilised for the detection of Hg²⁺ following the chemodosimetric approach with consequent change in the photophysical properties. The new reagent 127 having the λ_max of 319 nm was found to shift to 326 nm on reaction with Hg²⁺ due to the formation of HgSe (Fig. 75A).¹¹⁸ However, the chemical reaction was probed by monitoring the ∼100-fold enhancement in emission intensity with consequent increase in quantum yield from 0.002 to 0.2. This chemodosimetric reagent 127 could even detect Hg²⁺ as low as 0.18 ppb.

Fig. 75 (A) Mechanism showing removal of selenide atom due the action of Hg²⁺, which led to change in photophysical properties [inset: change observed under UV-light (a) only 127, (b) 127 in presence of Hg²⁺]. (B) Iridium-based chemodosimeter for the recognition of Hg²⁺ ions [inset: change in colour of 128 upon addition of Hg²⁺ under UV radiation]. Reprinted with permission from ref. 119. Copyright 2012 Royal Society of Chemistry.

A cyclometallated Ir(III) complex derived from benzimidazole moiety (128) could also detect Hg²⁺ with high specificity and sensitivity utilizing the change in optical and electrochemical properties (Fig. 75B).¹¹⁹ This Ir(III)-complex (128) exhibited two distinct absorption bands: band at 300–400 nm was assigned as a ligand based spin allowed¹ (π–π*) transition, while the other band at 400–525 nm was assigned to a MLCT and spin–orbit coupling enhanced³ (π–π*) transition. Addition of Hg²⁺ ions into the solution of 128 caused a lowering of the intensity of MLCT band, while gradual but subsequent increase in the intensity for the π–π* transition band was observed. This resulted in a change in solution colour from yellowish-green to colourless and the ratiometric spectral responses allowed probing the [Hg²⁺] in solution. A decomposition of the Ir(III)-chelate with the rupture of the relatively weak Ir–O bond was induced by Hg²⁺ ion without the compulsory involvement of a pre-coordinational step between the lone-pair electrons on the ligand and Hg²⁺. This was further confirmed from NMR and ESI-MS spectroscopy. Electrochemical studies revealed that the Ir^III/IV-based redox potential of 0.44 V for 128 was found to shift to 1.02 V on reaction with one equivalent of Hg²⁺, which also supported the decomplexation phenomena.

Hg²⁺ could also induce the desulphurization of thioxanthen-9-thione derivatives (129a and 129b) (Fig. 76) with associated changes in visually detectable change in solution colour from orange to colourless.¹²⁰ The absorption band for 129a was shifted from 487 to 397 nm in CH₃CN–H₂O medium (1 : 1, v/v), while significant luminescence changes were also observed for the transformation of thioxanthen-9-thione to thioxanthen-9-one. Detectable lowest concentration for Hg²⁺ was reported as 21 nM for the reagent 129a.

Fig. 76 Fluorescence changes in the presence of Hg²⁺ and proposed sensing mechanism: desulfurization of 129a and 129b, promoted by Hg²⁺ ions. Reprinted with permission from ref. 120. Copyright 2012 Royal Society of Chemistry.

Chemodosimeter 130 revealed a ‘turn on’ response in presence of Hg²⁺ in pure aqueous medium due to facile desulphurization–lactonization cascade transformation for the generation of highly fluorescent product from the non-fluorescent one (Fig. 77A).¹²¹ This resulted a ‘turn-on’ luminescence response with 50-fold enhancement of the band intensity at ∼480 nm only in presence of Hg²⁺ (λ_ext = 430 nm) within 30 s. Reaction stoichiometry evaluated for this process was 1 : 2, and the lower detection limit for Hg²⁺ was reported as 1 × 10⁻⁸ M⁻¹ considering the signal-to-noise ratio of 3.

Fig. 77 (A) Mechanism of Hg²⁺-mediated desulphurization–lactonization of 130. (B) Hg²⁺ recognition mechanism of 131.

Liu and Wang et al. developed a new ratiometric and chemodosimeter (131, Fig. 77B) for Hg²⁺ using chemoselective Hg²⁺-promoted desulfurization of a thioether to form an extended conjugated fluorescent system as a design strategy.¹²² In the absence of Hg²⁺, 131 displayed the coumarin-based emission at 488 nm on excitation at 435 nm in 0.1 M phosphate buffer (containing 0.5% acetonitrile) at pH 7.4. In presence of 2.0 equiv. of Hg²⁺, the emission at 488 nm disappeared and a new emission peak appeared at 560 nm, and the absorbance maxima was switched from 400 nm to 460 nm. This red shift in the absorbance and emission spectra and the ratiometric emission responses were due to the Hg²⁺ assisted desulfurisation of 131, which resulted in an α,β-unsaturated system, i.e. the extended conjugation in the fluorophore. The probe could even produce a pronounced fluorescent signal change when Hg²⁺ was as low as 2 × 10⁻⁸ M. Moreover, 131 could be used as intracellular fluorescent imaging reagent for the detection of Hg²⁺ in living cells.

Kim and co-workers synthesized two squarylium dye-based receptors 132 and 133 (Fig. 78).¹²³ Interestingly, these sensors could detect Hg²⁺ in DMSO medium following two different mechanisms. Coordination of Hg²⁺ to 132 caused desulphurisation with blue shift in absorption spectra from 659 nm to 625 nm, while for 133 the absorption spectra at 683 nm gradually disappeared with development of a new spectral band at 562 nm. Isosbestic point at 615 nm confirmed that Hg²⁺·133 was present in the equilibrium, and a stable 1 : 2 complex formation was proposed (Fig. 78). Thus, 132 is a chemodosimeter while 133 is a chemosensor.

Fig. 78 Representation of the proposed reaction of Hg²⁺ with reagents 132 and 133.

In most cases, Hg²⁺ leads to desulphurization and this leads to the perturbation of the energies of the frontier orbitals (HOMO and LUMO) with consequential changes in absorption and emission spectral pattern. This basic principal was utilized as receptor by Xiao, Qian and their co-workers for designing a new chemodosimeter (134) for Hg²⁺ ion (Fig. 79).¹²⁴ Reaction of the Hg²⁺ with the thiourea fragment led to the formation of oxadiazole derivative with associated opening of spirolactam ring of the rhodamine moiety, and this eventually led to the generation of new absorption band 560 nm along with BODIPY-based absorption at 501 nm in ethanol–water medium (4 : 1, (v/v); pH 7.0). This phenomenon helped in using this reagent as colorimetric reagent for detection of Hg²⁺ in aqueous sample.

Fig. 79 Hg²⁺-induced oxadiazole formation leading to the FRET process from BODIPY to rhodamine [inset: confocal images; (a) MCF7 cells incubated with 134 (5 μm) for 30 min at room temperature, emission measured at (514 ± 15) nm; (b) and (c) MCF7 cells incubated with 134 (5 μm), and then further incubated with Hg²⁺ ions (5 μm), emission measured at (514 ± 15, (b)) nm and (589 ± 15, (c)) nm]. Reprinted with permission from ref. 124. Copyright 2008 Wiley.

Using a similar strategy, Xiao and Qian and their co-workers had synthesized other FRET-based sensors, 135 and 136, which incorporated BODIPY as donor fragment and tetramethylrhodamine (TMR) unit as acceptor (Fig. 80) for the detection of Hg²⁺.¹²⁵ BODIPY–rhodamine platform (BRP) showed reversible switching of a rhodamine dye between a zwitterion and a lactone as a function of the media polarity and proticity.

Fig. 80 Molecular structures of the reagents 135 and 136.

In an aprotic solvent, such as acetonitrile, BRP emitted a strong green fluorescence with λ_max of 510 nm typical for BODIPY unit. In a protic solvent (methanol), BRP showed an orange fluorescence with λ_max of 568 nm, which is characteristic for rhodamine fragment even upon excitation of BODIPY unit at 488 nm. This clearly demonstrated that an effective FRET was switched on in methanol. Note that the rhodamine unit existed in the form of a ring-opened zwitterion in protic solvent like methanol that had a characteristic absorption peak at 540 nm, which was suited for inducing the FRET process. Emission spectral studies reveal that the BODIPY based emission at 514 nm was found to bleach gradually with simultaneous growth in emission intensity of new xanthene form of rhodamine-based band at 589 nm upon reaction with increasing amount of Hg²⁺ following a FRET process (with λ_ext 488). The FRET efficiency calculated for 136 was 99%, in which the distance between donor–acceptor was 58.9 Å. The fluorescence images of the MCF-7 cells were observed under confocal microscopy with the double channel fluorescence images at (514 ± 15) nm and (589 ± 15) nm in presence and absence of HgCl₂. Observed change in emission signal from green to red further confirmed that the FRET process was operational.

A chromofluorogenic dual functionalised hybrid material (137) was made of mesoporous material with homogeneously distributed pores having ∼2 to 3 nm in size with specific surface area of 1000 m² g⁻¹ for the detection of Hg²⁺ in aqueous environment. The nano-sized silica particles were covalently linked to the thiol functionality as the binding site by silylation reaction and thiols groups are also covalently linked to the signalling unit, squarine dye.¹²⁶

The actual strategy was designed in such way that Hg²⁺ would react readily with thiol functionality with release of the highly coloured and emissive squarine dye, as shown in Fig. 81. This probe could allow detection of Hg²⁺ as low as ppb, while the solid material could absorb Hg²⁺ for the concentration range of 0.7–1.7 mmolg⁻¹ (depending on the degree of the functionalization) in acetonitrile–water medium (1 : 1, v/v, pH 3). Thus, this material could be used as Hg²⁺ ion sponge with fluorescence responses.

Fig. 81 Protocol used for the detection and removal of Hg²⁺ from aqueous environment.

Water soluble CdSe–ZnS-based QDs were functionalized with 2-hydroxyethyl dithiocarbamate and these modified QDs showed ability to detect Hg²⁺ from its aqueous solution (Fig. 82).¹²⁷ Preferential binding of the thiol functionality to Hg²⁺ led to the leaching of the 2-hydroxyethyl dithiocarbamate moiety from the CdSe–ZnS-based QD surfaces with associated change in solution colour from orange to red with the detection limit of 1 ppb. The corresponding shift in emission maxima was from 596 nm to 603 nm. The quantum dot-based sensors were also utilised on cellulose acetate paper. A visually distinguishable fluorescence (from orange to red) could be clearly observed depending on the concentration of Hg²⁺, to which these modified cellulose papers were exposed. The lowest detection limit obtained in this method was 0.2 ppb.

Fig. 82 Quantum dot-based Hg²⁺ sensors [inset: colour change observed on cellulose acetate paper on dropping solution of Hg²⁺]. Reprinted with permission from ref. 127. Copyright 2012 American Chemical Society.

Triboelectric nanogenerator for the Hg²⁺ recognition

Apart from these conventional colorimetric, fluorometric sensors, a new kind of recognition phenomenon was observed by Wang and his co-workers.^128,129 They demonstrated an innovative and unique approach toward self-powered detection of Hg²⁺.

They developed the first triboelectric nanogenerator (TENG) effect-based highly sensitive and selective sensor for the detection of Hg²⁺ ions by using 3-MPA (mercapto propionic acid)-modified AuNPs as electrical performance enhancer and recognition element. Based on the high power density (6.9 mW cm⁻²) of this as-developed TENG, a commercial LED lamp can be used as an indicator instead of expensive electrometers (Fig. 83). This novel TENG-based sensor system is quite sensitive (detection limit of 30 nM and linear range of 100 nM to 5 μM) and selective for the detection of Hg²⁺ ions. With its high sensitivity, selectivity and simplicity, TENG holds great potential for the determination of Hg²⁺ ions in environmental samples.

Fig. 83 Rectified J_sc of the as-developed TENG before (a) and after (b) the interaction with 5 μM Hg²⁺ ions. Insets: photograph of the indicated LED lamp before (a) and after (b) interaction with 5 μM Hg²⁺ ions, as an indication of detected concentration. Reprinted with permission from ref. 129. Copyright 2013 Wiley.

Conclusion

For Hg(II), the “heavy atom” effect favours an enhanced spin–orbit coupling constant (ζ), and generally induces a strong luminescence quenching of the bound luminophore. This together with the high hydration enthalpy (1824 kcal mol⁻¹) for Hg²⁺ ion poses a challenge to the researchers for achieving the seemingly simple but tricky issue of the Hg²⁺ recognition with luminescence on or enhancement response either in aqueous environment or in physiological conditions. Luminescence enhancement/on response is crucial for designing reagent for imaging application for detection of the cellular uptake of this ion. This is significant in developing efficient reagent for diagnostic application and environmental sample analysis, in addition to addressing the other crucial issue of sensitivity. Furthermore, specific detection of Hg²⁺ in presence of other soft transition metal ions like Cu²⁺, Cd²⁺, Pb²⁺ and Ag⁺ remains a challenge for researchers. In this report, we have summarized a brief but extensive account of all such reports along with the reports on colorimetric reagent that are conducive for in-field sample analysis with yes–no type binary response. Attempts to develop self-indicating solid surfaces for Hg²⁺ detection and scavenging are also summarized in this article.

Acknowledgements

AD thanks DST (India) and CSIR (India) for financial support. HA acknowledges CSIR for his research fellowship.

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Footnote

† Authors have contributed equally.

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