Fluorescent, colourimetric, and ratiometric probes based on diverse fluorophore motifs for mercuric(II) ion (Hg²⁺) sensing: highlights from 2011 to 2019

Abstract

Though it has not been shown to deliver any biological importance, mercuric(II) ion (Hg²⁺) is a deleterious cation which poses grievous effects to the human body and/or the ecosystem, hence, the need for its sensitive and selective monitoring in both environmental and biological systems. Over the years, there has been a great deal of work in the use of fluorescent, colourimetric, and/or ratiometric probes for Hg²⁺ recognition. Essentially, the purpose of this review article is to give an overview of the advances made in the constructions of such probes based on the works reported in the period from 2011 to 2019. Discussion in this review work has been tailored to the kinds of fluorophore scaffolds used for the constructions of the probes reported. Selected examples of probes under each fluorophore subcategory were discussed with mentions of the typically determined parameters in an analytical sensing operation, including modulation in fluorescence intensity, optimal pH, detection limit, and association constant. The environmental and biological application ends of the probes were also touched where necessary. Important generalisations and conclusions were given at the end of the review. This review article highlights 196 references.

Introduction

Mercury (II) (Hg ²⁺ ) detection

Generating means to detect mercury(II) (Hg²⁺) is necessary since reports have shown Hg²⁺ to be a health threat to the world environment, this stemming from its highly toxic nature and ubiquitous impact in both the lithosphere and hydrosphere (Coronado et al. 2005). Years of experimental works have shown Hg²⁺ to pose health problems not only to human beings but also to other animal species. Hg²⁺ contamination is being engendered through natural and anthropogenic endeavours, including ocean and volcanic eruptions, mining of metal (especially artisanal and small-scale gold mining, ASGM), incineration of waste, tanning of leather, electroplating, occupational operations, dental care, preventive medical practices, agricultural and industrial operations, and fossil fuel combustion (from coal-burning power plants) (Renzoni et al. 1998; Rice et al. 2014; Bravo et al. 2014; Costa et al. 2016; Martinez-Finley et al. 2014). Elemental mercury and ionic mercury (Hg and Hg²⁺, respectively) have the tendency to be transformed by microorganisms like bacteria into methylmercury (MeHg), which is a potentially harmful neurotoxin capable of bioaccumulation into the food chain (Nolan and Lippard 2008). Its damaging effect on the placenta of pregnant women has been particularly identified, which thereby poses a developmental problem to foetal brain and central nervous system, resulting in a developmental delay in children (Coronado et al. 2005; Kim et al. 2008; Gibb and OLeary 2014). This can then implicate certain biological processes, inducing a chain of diseases including splanchnic damage, nosebleed, headache, perforation of the stomach, nerve disorder, intestines septum, and acute renal failure (Renzoni et al. 1998; Zhai et al. 2012; Hoyle et al. 2005; Zarlaida 2017). As such, exposure to low concentrations of Hg²⁺ can trigger numerous grievously damaging effects to the human heart, kidney, stomach, and intestines, resulting in metabolic disorders, motor and cognitive disorders, and sensory malfunctions and disorders (Rice et al. 2014; Hoyle and Handy 2005; Arya and Bhansali 2011; Ghasemi et al. 2018; Wang et al. 2008). This has resulted in stringent regulations imposed on relevant industries to bring about a clean-up system for mercury-laden wastewater before its discharge into waste streams. The maximum permissible limit of total mercury in wastewater discharge is 10 µg/L (US Environmental Protection Agency 2001; Kumar et al. 2012a). US EPA’s previously regularised guideline of Hg²⁺ in drinking water of 1 µg/L was changed to 6 µg/L in 2005—the former value being based on methylmercury (MeHg), whilst the recent guideline is based on inorganic mercury (Frisbie et al. 2015). Amongst the various heavy metals, Hg²⁺ ion shows the most toxicity, hence, the inspiration and need for its monitoring. These obvious environmental and biological threats which (Hg²⁺) poses have engendered the construction of methods for its detection and measurement in aqueous phases—three of such widely exploited concepts being the fluorescent, colourimetric, and/or ratiometric detection modes due to their pre-eminence over other detection techniques.

The fluorescence, colourimetric, and ratiometric optical detection methods

The past twenty plus years of the arrival of the supramolecular chemistry have recorded stunning successes in the constructions of fluorescent, colourimetric, and ratiometric probes as vibrant, vital tools for the sensing of environmentally and biologically relevant cations and anions, and also for the detection of H⁺ (proton) in biological assays (Gunnlaugsson et al. 2006; Lodeiro and Pina 2009; Duke et al. 2010; Georgiev et al. 2011; Park et al. 2020). Compared to conventional analytical detection methods of voltammetry, polarography, X-ray fluorescence analysis, infrared spectroscopy, atomic fluorescence spectrometry, high-performance liquid chromatography, mass spectrometry, atomic absorption spectroscopy, etc., which have been utilised to recognise heavy metal ions like Hg²⁺, the fluorescence method is a preferred, sought-after, and powerful analytical tool. The fluorescence technique has been employed for molecular recognition since it offers the benefits of high spatial resolution, ultra-high sensitivity and selectivity, real-time detection, local observation, operational simplicity, equipment’s inexpensiveness, quick response time, and non-destructive nature (Lee et al. 2015; Carter et al. 2014; Zhang et al. 2014; Kim et al. 2012, 2011; Chen et al. 2012; Yang et al. 2013; Singha et al. 2019). Because of these merits, there have been successful instances of the application of the fluorescent technique in clinical diagnostics, biotechnology, biochemistry and molecular biology, materials and environmental sciences (Mason 1999). It has been used for the monitoring of diverse groups of analytes including heavy metal ions, explosives, peptides, and pesticides (Bigdeli et al. 2019).

The colourimetric method, a powerful analytical technique like the fluorescence method, has also been harnessed in the field of sensing science as it affords the ability for naked-eye monitoring of analytes even before spectrophotometric analytical investigation (Kaur et al. 2018). Colourimetric probes upon interaction with analytes give ‘naked-eye’ (i.e. visibly detectable) colour changes, which are easily detectable signal outputs that do not need any sophisticated instruments. Diagnostic assays like blood-glucose monitoring and early pregnancy tests are two key instances where the colourimetric method has been successfully applied (Bicker et al. 2011).

The ratiometric detection approach has been put in place to overcome some sensitivity difficulties usually encountered in optical detection (e.g. interference from a variety of analyte-independent factors, including instrumental parameters, the probe’s microenvironment, the probe’s local concentration, and photobleaching) (Bigdeli et al. 2019; Lee et al. 2015). Ratiometric fluorescence detection method affords the possibility of simultaneous recording of two measurable signals in the presence and absence of analyte (Grynkiewicz et al. 1985). This detection technique is usually both desirable and preferable as it can measure fluorescence signals at two different wavelengths unlike the traditional methods that measure fluorescence intensity only at a single wavelength. Fluorescent resonance energy transfer (FRET) allows for the possibility of precise and quantitative read-outs. Due to this desirable property of FRET, many ratiometric fluorescent probes, such as some discussed in this review, have been designed by adopting the FRET-based ratiometric approach. FRET, in principle, is a distance-dependent physical process involving the non-radiative transfer of energy from an excited molecular fluorophore, i.e. the donor, to another fluorophore, i.e. the acceptor, through an intermolecular long-range, dipole–dipole coupling. FRET can measure molecular closeness at angstrom distances of 10–100 Å and is highly efficient if the donor and acceptor are positioned within the Förster radius (i.e. the distance at which half the excitation energy of the donor is transferred to the acceptor, usually 3–6 nm). FRET is a sensitive technique that could investigate biological processes which induce changes in molecular proximity as it is dependent on the one-sixth of inter-molecular separation (Förster 965; Clegg 1996; Lakowicz 1999; dos Remedios et al. 1987). Apart from FRET, in the context of this review work, other detection mechanisms/strategies including photoinduced electron transfer (PET), internal charge transfer (ICT), twisted intramolecular charge transfer (TICT), electron transfer (ET), energy transfer (eT), through-bond energy transfer (TBET), fluorescence resonance energy transfer (FRET), time-gated fluorescence resonance energy transfer (TG-FRET), aggregation-induced emission (AIE), through-bond fluorescence resonance energy transfer (TBFRET), etc., have also been employed for the design of some reported probes towards Hg²⁺detection.

Overview of commonly used molecular motifs for sensing operation

Prompted by the efficacy of the fluorescence, colourimetric, and ratiometric detection techniques, a plethoric number of probes have been developed using various categories of fluorophore motifs. The analytical advantages of small-molecule probes have been recognised, viz high selectivity, bioorthogonality, minimal perturbation to living systems, high sensitivity, simplicity of manipulation, and non-sophistication of instrumentation (Chan et al. 2012; Chen et al. 2011). The year 1845 saw the report of the first fluorescent organic molecule, the sulphate of quinine, which was discovered by Sir John Herschel (1845), whilst the year 1852 saw the advent of the term ‘fluorescence’, which was conceptualised by Stokes (1845). The discovery of quinine sulphate (Fig. 1) has informed the discovery of many other organic compounds which fluoresce with distinctive colours. These include fluorescein and rhodamine derivatives, boron-dipyrromethene (BODIPY) dyes, cyanine dyes, and others (Fig. 1) (Zheng et al. 2013; Baeyer 1871; Ceresole 1888; Loudet and Burgess 2007; Luo et al. 2011). Dyes which fluoresce with red to near-infrared (NIR) light show great potentials for in vivo imaging in biological systems, owing to their ability to penetrate tissues in the red to near-infrared wavelength region (650–900 nm) (Luo et al. 2011; Weissleder and Ntziachristos 2003). This has implied the development of a new class of dyes, which not only emit red or NIR fluorescence but possess higher extinction coefficients. This class of dyes anchors fluorophores functionalised with additional ring systems or heteroatoms or dye moieties which have been structurally modified. (Umezawa et al. 2008; Yuan et al. 2012; Egawa et al. 2011; Kim et al. 2011; Samanta et al. 2010). Lately, fluorescent dyes with two-photon imaging and super-resolution imaging abilities (Kim and Cho 2009; Krishna et al. 2006; Maçôas et al. 2011) have been gaining momentum for sensing purposes.

Fig. 1

Structures of some common organic compounds employed as small-molecule probes (Herschel Sir 1845; Zheng et al. 2013; Baeyer 1871; Ceresole 1888; Loudet and Burgess 2007; Luo et al. 2011)

Probes based on supramolecular structures usually show superior sensing properties to those based on simple synthetic organic dyes. Common supramolecular structures which are successful in the designs and syntheses of Hg²⁺ probes include 1,4,7,10-tetraazacyclododecanes (cyclen), cyclodextrin, calixarene, porphyrin, crown ethers, helicene, peptidyl, amongst others (Fig. 2) (Kappe 1994; Liu et al. 2008; Lee et al. 2018; Li et al. 2018; Shen and Chen 2012; Tung 2004; Athey and Kiefer 2002; Rothemund 1936; Gierczyk et al. 2013). Many synthetic organic probes have been simply employed in their polymeric forms for improved sensing properties. Polymer-embedded optical probes stand out for their several merits, including, signal amplification, ease of use, simplicity of fabrication into devices, and combination of different outputs (Kim et al. 2011).

Fig. 2

A few examples of common supramolecular structures employed as probes (Kappe 1994; Liu et al. 2008; Lee et al. 2018; Li et al. 2018; Shen and Chen 2012; Tung 2004; Athey and Kiefer 2002; Rothemund 1936; Gierczyk et al. 2013; Urbano 2003)

A burgeoning interest exists in the development of probes based on metal-organic frameworks (MOFs) and covalent-organic frameworks (COFs) as they possess outstanding features of large surface areas, regular pore structures, tunability in their pore sizes and functionalities, and thermal stabilities (Zhang and Yuan 2018; Rudd et al. 2016). These strong merits make them employable as probes for imaging applications. Typically, MOFs have two components: the metal ion or cluster and the organic linker. Common organic linkers used for the constructions of MOFs have been summarised in a paper published by Allendorf’s group. The properties of MOFs can be modulated easily by varying the linker, metal, and/or growth conditions (Allendorf et al. 2009). Covalent organic frameworks (COFs) are covalent porous crystalline polymers that enable the elaborate integration of organic building blocks into an ordered structure with atomic precision. COFs possess high thermal stabilities, low mass densities, and long-lasting porosity. These interesting properties of COFs have made them useful towards sensing ends. Both two-dimensional (2D) and three-dimensional (3D) COFs have been recognised (Feng et al. 2012). A diversity of building blocks is available for the development of COFs, and this has been given in a reported literature (James 2003).

Research investigations have found out that some drawbacks of fluorescent dyes can be combated or at the very least ameliorated by the enclosure of the dye in nanoparticle delivery systems (Koo et al. 2007). The distinctive optical and electronic properties of nanomaterials have informed their increasing use as probes and diagnostic agents (Xu et al. 2014). Probes based on nanomaterials possess the advantages of stronger fluorescent emission, large surface-area-to-volume ratio, stability, multifunctionality, target specificity, simplicity, novelty, high selectivity and sensitivity, excellent sensing parameters such as low detection limit and water solubility (Guo et al. 2014; Song et al. 2010). Common examples of nanomaterials include gold nanorods, silver nanoparticles, DNA oligomers, silica nanoparticles (Xu and Suslick 2010; Guo et al. 2009; Ritchie et al. 2007; Shang and Dong 2008; Zhou et al. 2016; Zhang et al. 2015a), quantum dots (QDs) (e.g. CdSe and CdTe), TiO₂ NPs, SiO₂ NPs, and Fe₃O₄ NPs (Lin et al. 2011). Probes based on QDs show superior features of outstanding stability, tunable and minuscule emission spectrum, large quantum yield and Stokes’ shifts, wide excitation wavelength, which mirror the merits of synthetic organic probes. Graphene oxides (GOs) show tunable physicochemical properties and good dispersion ability in many solvents, especially in water (Freeman et al. 2009; Abdelhamid and Wu. 2015). Many mercuric (II) (Hg²⁺) nanoprobes either make use of the change in colour (absorption) or fluorescence intensity (‘turn on’ and ‘turn off’) in their detection modes. Some of these can be DNA-functionalised or non-DNA-functionalised.

The purpose, scope, and structure of this current review

To be as brief and direct as possible, the fundamental rules and principles governing the design strategies of probes are left out in this review paper. Insights into these have been given in some other excellent review works (Chan et al. 2012; Kaur et al. 2016). Even though as of the present, a large assembly of fluorescent, colourimetric, and/or ratiometric probes for Hg²⁺-selective monitoring with suitable applications have been covered in a few previously reported works; however, such exist in rather ‘old’ references. (Readers are referred to see the review articles by Nolan and Lippard (2008), Mahato et al. (2014), and Chen et al. (2015.) This current review has the purpose of presenting a detailed account of the recent progress made in the designs and syntheses of ‘new’ optical probes for Hg²⁺ detection from 2011 to 2019.

This article is structured in such a way that the body of the review work is based on four broad categories of selected fluorophore scaffolds used for the structural designs of the probes reported (Fig. 3): 1. simple molecular ligands/synthetic organic dyes; 2. supramolecules: carbohydrates and polymers; 3. metal-organic frameworks (MOFs) and covalent organic frameworks (COFs); and 4. nanoprobes: nanoparticles, quantum dots (QDs), and graphene oxides (GOs) (Fig. 3). The sensing properties of the probes under each subsection have been discussed by emphasising the parameters of the most suitable solvent system, optimum pH value/range, interference property, detection limit, and association constant. Some occasional mentions of the probes’ utility in imaging Hg²⁺ in biological systems are also given. Sufficient references, totalling 196 articles, are reviewed in strength to keep abreast of the works done in various labs whilst ensuring succinctness of the report. All reported compounds either employ the fluorescence, colourimetric, and/or ratiometric method in their detection modes (Fig. 3). In some instances, however, the mentioned compounds make use of combined fluorescence or colourimetric or ratiometric methods in their sensing technique/detection methodology. This review closes by giving a brief conclusion that will help guide future research works in the chemistry of Hg²⁺ sensing.

Fig. 3

The future of Hg²⁺ sensing science is bright with key classes of commonly employed fluorophores and signalling methodologies recognised. Source: the author

Hg²⁺-selective small-molecule probes based on different fluorophore motifs

Based on different subfluorophores, an account of small-molecule probes for the monitoring of mercuric (II) ion (Hg²⁺) is detailed below.

Small-molecule Hg ²⁺ probes based on simple molecular ligands/synthetic organic dyes

A large percentage of probes that exist to date are based on conventional simple molecular ligands/synthetic dyes constructed from various organic fluorophores such as the 1,8-naphthalimide, rhodamine, coumarin, pentaquinone, boron-dipyrromethene (BODIPY), cinnamaldehyde, Schiff base, anthracene, squaraine, pyrene, azo derivative, amongst many others. Small-molecule probes do not require invasive and often tedious genetic modification of the system and can be applied to many samples, and they, therefore, demonstrate potential clinical applications (Kim and Cho 2015).

Small-molecule Hg ²⁺ probes based on naphthalimide fluorophore

The naphthalimide fluorophore possesses several desirable properties including a large Stokes’ shift and a high quantum yield that inform its choice as a common fluorophore for the development of probes (Aderinto et al. 2016; Aderinto and Imhanria 2018; Wang et al. 2017). Moreover, its chemistry is versatile, allowing the possibility for distinct functionalisation that confers tailored properties on the probes constructed from this moiety (Moro et al. 2010).

Chen’s group recently reported a naphthalimide-based fluorescent and colourimetric probe, NAP-PS, that houses the diphenylphosphinothioyl group for Hg²⁺ detection (Chen et al. 2019). In HEPES buffer (1.0 mM, pH = 7.4), amongst the many other ions (Zn²⁺, Cu²⁺, Mn²⁺, Pb²⁺, Al³⁺, Ca²⁺, Cr³⁺, K⁺, Fe³⁺, Fe²⁺, Cs⁺, Na⁺, Ni²⁺, Mg²⁺, Sr²⁺, Cd²⁺, and Ag⁺) investigated, only the addition of Hg²⁺ leads to a significant ratiometric response of the fluorescence intensities at 450 nm to 550 nm. The ratiometric fluorescence response of NAP-PS is due to its reaction with mercury, which cleaves the thiophosphinate P–O bond in the probe to form the intramolecular charge transfer (ICT) system, NAP-O. The linear range of detection was within 0–12 nM, whilst the detection limit was calculated as 43 nM. The probe was also capable of Hg²⁺ monitoring in the solid state. Using two-photon microscopy, the designated probe could bioimage Hg²⁺ in both HeLa live cells and tissues with very low cytotoxicity exhibited.

La’s research group reported the design of their newfound 4-Amino-1,8-naphthalimide-based fluorescent probe, Hg-1 (Fig. 4), for Hg²⁺ monitoring, which could also sense CN⁻ and F⁻ via the colourimetric method, thereby acting as a multianalyte probe (La et al. 2016). The design strategy was based on the combination of both PET and ICT, which allow for a dual analyte monitoring mode. (PET-based probe design involves the fluorophore unit connected to the ionophore unit through a spacer, whilst ICT-based probe design involves the direct connection of the fluorophore unit and ionophore unit to form a single species (Gupta and Kumar 2016.) The reported compound underwent a multiplication in its fluorescence signal intensity after Hg²⁺ was added, whilst the other ions tested together did not modulate the fluorescence signal intensity of the probe significantly. There was a linear response of the probe for Hg²⁺ monitoring with the detection limit reportedly low to the level of 2.40 × 10^–7 M (lower than those of previously reported probes), and the association constant reportedly high as 4.12 × 10⁵ M. The analysis of the Job’s plot and ¹H NMR results gave the stoichiometric ratio to be 1:1 (Fig. 5).

Fig. 4

Structures of Probes Hg-1, Hg-2, and Hg-3 (La et al. 2016; Li et al. 2016)

Fig. 5

Proton NMR of Probe Hg-1 in the absence and presence of excess Hg²⁺. Key finding: there was an upfield shift in the aromatic protons owing to increased electron density upon protonation (La et al. 2016)

Two naphthalimide-appended fluorescent probes, Hg-2 and Hg-3 (Fig. 4), were devised and reported by Li and his research group members (Li et al. 2016). In phosphate buffer, the two compounds display ultra-high-level sensitivities and selectivities for Hg²⁺ detection. It was observed that Hg-2 and Hg-3 could detect Hg²⁺ over a vast expanse of pH, between 7.0 and 10.0. In a 10 μM solution of Hg-2 in phosphate buffer (pH = 7.5), upon the addition of cations like Ag⁺, Al³⁺, Ba²⁺, Cd²⁺, Ca²⁺, Co²⁺, Cr³⁺, Cu²⁺, Fe²⁺, Li⁺, K⁺, Na⁺, Mg²⁺, Zn²⁺, Mn²⁺, Pb²⁺, Ni²⁺, and Hg²⁺, only Hg²⁺ led to ca. 90% suppression of the fluorescence intensity of Hg-2; meanwhile, the other cations impressed only benign effects on the fluorescence intensity. A similar observation went for Hg-3. There was linearity in Hg²⁺ monitoring by Hg-2 between the concentration range of 2 and 10 μM, and the lowest detection limits of Hg-2 and Hg-3 for Hg²⁺ detection were 2.1 μM and 3.1 μM, respectively. Analytically, Hg-2 exhibits higher selectivity and sensitivity towards Hg²⁺ than Hg-3, this being accrued to the fact that the conjugation action of morpholine which exists in Hg-3 makes it to poorly bind to Hg²⁺. The proton NMR of the binding of Hg-2 to Hg²⁺ shows the disappearance of a major peak downfield in the NMR spectrum upon the addition of Hg²⁺.

Work in mercury probes’ development has also shifted towards the employment of sulphur-based functional groups (like sulphoxides and thioureas) since they display great affinities for measured analytes in aqueous environments. Vonlanthen and his team members developed a PET thiourea–naphthalimide-based probe, Hg-4 (Fig. 6) that demonstrates Hg²⁺ sensing over competing metal ions like Na⁺, K⁺, Mg²⁺, Ca²⁺, Ag⁺, Zn²⁺, Cd²⁺, Hg²⁺, and Pb²⁺ in CH₃OH (Vonlanthen et al. 2014). At pH 5.5 or lower, in 9:1 H₂O/CH₃OH, no fluorescence enlargement was observed, but there was a massive amplification of the fluorescence in the case of Hg²⁺. Job’s plot establishes a 1:1 stoichiometry of the interaction between the probe and Hg²⁺ ion. Eventually, the compound was applied to image Hg²⁺ in living mammalian cells (Fig. 7), indicating its practical utility potential in biological system.

Fig. 6

Scheme of fluorescent Probe Hg-4 exhibiting the PET mechanism (Vonlanthen et al. 2014)

Fig. 7

Fluorescence images showing HeLa cells treated with Probe Hg-4 and HgCl₂. a HeLa cells treated overnight with a DMSO control at 37 °C. b Cells treated overnight with a DMSO control followed by HgCl₂ (200 μM) for 10 min at 37 °C. c Cells incubated overnight with Probe Hg-4 (20 μM) at 37 °C. d Cells treated overnight with Probe Hg-4 (20 μM) followed by HgCl₂ (200 μM) for 10 min at 37 °C. Imaging was done on a Zeiss Axio Observer inverted microscope with objective = ×63; excitation wavelength = 365 nm, emission wavelength = 445/50 nm (Vonlanthen et al. 2014)

Un et al. introduced a novel, simple, and versatile fluorescent Probe Hg-5 (Fig. 8) based on the N-butyl-4-bromo-1,8-naphthalimide moiety. The probe was employed for a fluorescence ‘turn on’ monitoring of Hg²⁺ over a wide range of several other cations, including Zn²⁺, Mn²⁺, Ba²⁺, Ni²⁺, Cu²⁺, Co²⁺, Pb²⁺, Mg²⁺, Cd²⁺, Fe²⁺, Al³⁺, Ag⁺, Na⁺, and Li⁺ (Un et al. 2014). In aqueous solution (THF-H₂O, 1:1, pH = 7.4, 10 mM Tris–HCl), Hg²⁺ addition to a solution of Hg-5 induced a notable enhancement of fluorescence. The fluorescence detection occurred over the range 1–30 μM, with a detection limit of 6.28 × 10^–8 M and an association constant of 5.4 × 10⁴ M⁻¹ calculated. The fluorescence imaging experiments of Hg-5 for Hg²⁺ tracking in living cells were successfully demonstrated.

Fig. 8

Structure of Probe Hg-5 (Un et al. 2014)

Moon et al. employed a new thionaphthalimide-based probe, Hg-6, and its two monothio derivatives, Hg-7 and Hg-8 (Figs. 9, 10), for the sensitive and selective monitoring of Hg²⁺ through an ‘off–on’ mode (Moon et al. 2013). In 30% aqueous CH₃CN, the fluorescence intensity of Probe Hg-6 at 537 nm was escalated upon the addition of 20 equivalents of Hg²⁺, whereas the effects of other tested cations on the fluorescence intensities of the probes were minuscule. The reported Probe Hg-6 could allow the detection of Hg²⁺ ion in an amount as low as 2.7 μM. The isomeric probes Hg-6 and Hg-7 were demonstrated to exhibit varied sensing behaviours with Hg-6 showing a faster signalling speed than Hg-7. This was explained as due to the carbon resonance of the more electron-rich thiocarbonyl group of Hg-6 which possesses more electron-rich sulphur and appears in a higher field (192.5 ppm) than that of Hg-7 which appears in a lower field (194.2 ppm) (Fig. 9). The signalling behaviour of the dithio-derivative, Hg-8, was not reported, only that upon interaction with Hg²⁺, it became desulphurised to the monothiol-derivative, Hg-7.

Fig. 9

Partial ¹³C NMR spectra of Probes Hg-6 and Hg-7 in CDCl₃ (Moon et al. 2013)

Fig. 10

Structure of Probe Hg-8 (Moon et al. 2013)

Probe Hg-9 (Fig. 11) was used by Zhang et al. for the signalling detection of Hg²⁺ amongst several other cations, including Na⁺, K⁺, Ca²⁺, Mg²⁺, Cu²⁺, Zn²⁺, Cr²⁺, Pb²⁺, Ni²⁺, Fe²⁺, Mn²⁺, Co²⁺, and Cd²⁺ (Zhang et al. 2013). In EtOH/H₂O (1/2, v/v), the addition of Hg²⁺ to a solution of the probe led to a marked increase in the fluorescence intensity of Hg-9, whilst no obvious disturbance in detection was observed from other tested cations. The binding stoichiometry of Hg-9 towards Hg²⁺ as furnished by the Job’s plot analysis yielded the establishment of a 1:1 complex between Hg²⁺ and Hg-9. This was further evidenced by the results of the proton NMR.

Fig. 11

Structure of Probe Hg-9 showing its binding mode with Hg²⁺ (Zhang et al. 2013)

Li et al. reported a PET naphthalimide-based probe, Hg-10 (Fig. 12), that bears a hydrophilic hexanoic acid group (as a solubilising functional group) for the monitoring of Hg²⁺ (Li et al. 2012). Experimental results revealed that the compound was most effective for the tracking of Hg²⁺ within the linear concentration range of 2.57 × 10⁻⁷–9.27 × 10⁻⁵ M. Job’s plot unveiled a 1:1 stoichiometric mode of Probe Hg-10 and Hg²⁺. The calculated detection limit was 4.93 × 10^–8 M. In Tris-HNO₃ buffer solution (pH = 7.0), the probe showed a massive enhancement in the intensity of its fluorescence upon Hg²⁺ addition in coexistence with several other investigated cations. The analytical detection response time of Hg²⁺ by Hg-10 was < 1 min. Hg-10 exhibited a regeneration ability in its sensing mechanism as ascertained by the release of the unbound probe upon the addition of EDTA to the Hg-10/Hg²⁺ complex. The probe was successfully used for Hg²⁺ monitoring in hair samples.

Fig. 12

Structure of Probe Hg-10 (Li et al. 2012)

Small-molecule Hg ²⁺ probes based on rhodamine fluorophore

Since they possess excellent photophysical properties, rhodamines have enjoyed wide applications as fluorescent probes and molecular markers. Rhodamine dyes, which belong to the xanthene class of dyes, represent one of the oldest synthetic dyes used for fabric dyeing. They possess high molar absorptivities in the visible region, and many of their derivatives exhibit strong fluorescence. Moreover, there is a strong influence of their absorption and emission properties by substituents in the xanthene nucleus, thereby making them useful not only as colourants but also as fluorescent markers in photosensitisers, microscopic structural studies, and laser dyes (Arbeloa and Ojeda 1981).

Arivazhagan et al. designed two triazole-appended ferrocene–rhodamine conjugates, C₄₇H₄₅N₇O₃Fe (Hg-11) and C₄₉H₄₉N₇O₃Fe (Hg-12) (Fig. 13), synthesised as probes for a highly ‘turn on’ fluorescence detection of Hg²⁺ in CH₃CN/HEPES buffer (2:8, v/v, pH 7.3, 10 μM) (Arivazhagan et al. 2015). It was shown that receptors Hg-11 and Hg-12 could be employed for Hg²⁺ monitoring in the near-neutral pH range of 7.3. Upon the addition of Hg²⁺ to the receptors Hg-11 and Hg-12, there was a massive enlargement of the fluorescence intensity, explained based on the spirolactam ring opening of the rhodamine moiety upon chelation with a cation. The addition of alkali, alkaline, transition, and heavier transition metal ions like Na⁺, Mg²⁺, K⁺, Ca²⁺, Cr²⁺, Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Pb²⁺, Cd²⁺, and Tl⁺ effected almost no influence on the fluorescence emission spectrum of receptor Hg-11, which was further confirmed by competitive experiments, thereby demonstrating the high affinity of receptor Hg-11 for Hg²⁺. The same trends of observations went for receptor Hg-12. Desirably, experimental results revealed that the receptors were reversible in their monitoring of Hg²⁺. Since the receptors could image Hg²⁺ ion in living cells using the MTT (5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) assay without any destruction, their potencies to track intracellular Hg²⁺ ions in MCF-7 breast cancer cells were successfully assessed via fluorescent imaging studies (Fig. 14).

Fig. 13

Structures of Probes Hg-11 and Hg-12 (Arivazhagan et al. 2015)

Fig. 14

Fluorescence and bright-field images of MCF-7 cells. a Fluorescence image of MCF-7 cells incubated with Hg-11 (10 μM) for 2 h at 37 °C (left) or Hg-12 (10 μM) for 2 h at 37 °C (right). b Bright-field image of Hg-11-treated MCF-7 cells (left) or Hg-12-treated MCF-7 cells (right). c Fluorescence image of MCF-7 cells incubated with Hg-11 (10 μM) for 30 min and then treated with (10 μM) Hg(ClO₄)₂ for 2 h at 37 °C (left) or Hg-12 (10 μM) for 30 min and then treated with (10 μM) Hg(ClO₄)₂ for 2 h at 37 °C (right). d Overlaid images of MCF-7 cells (b, c) (left: Hg-11; right: Hg-12) (Arivazhagan et al. 2015)

Chen and members of his research laboratory reported a water-soluble fluorescent probe, Hg-13 (Fig. 15), constructed from rhodamine B derivative (Chen et al. 2013b). The new probe displayed both colourimetric (‘naked eye’) detection and fluorescent response towards Hg²⁺ in a fully aqueous HEPES buffer solution. The optimum experimental pH range for the sensing of Hg²⁺ by Hg-13 was found to be 6–11. At 586 nm, a sizeable fluorescent escalation of about 1500-fold increment was noted upon Hg²⁺ addition to the solution of the probe in the presence of other alkali, alkaline earth, and transition metal ions (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cr³⁺, Fe³⁺, Fe²⁺, Cd²⁺, Mn²⁺, Pb²⁺, Zn²⁺, Cu²⁺, Ag⁺, Co²⁺, Ni²⁺) investigated under identical testing conditions. Association constant of 1.21 × 10⁵ M⁻¹ was estimated from the binding interaction of the probe and analyte metal ion. Besides the linearity of the relationship between the probe and Hg²⁺, Job’s plot yielded a 1:1 stoichiometric ratio of the probe and the metal ion in the Hg-13/Hg²⁺ complex. The reversibility nature of the probe was demonstrated after the addition of EDTA to the complex’s solution, which led to the release of free Hg-13 compound from the bound Hg-13/Hg²⁺ complex. A fast response time (there was no apparent time delay) was demonstrated in the tracking of Hg²⁺ by Hg-13 as justified by the results of the real-time analysis. Utilising the fluorescence imaging experiments, the biological utility of Hg-13 demonstrated its ability to image Hg²⁺ in living HeLa cells whereby an MTT assay gave cell viability of 95% after treatment with 10 μM of the compound after 24 h of incubation.

Fig. 15

Structures of Probes Hg-13, Hg-14, and Hg-15 (Chen et al. 2013b; Gong et al. 2012; Hu et al. 2013)

For fluorescence detections of cations, it has been purported that virtually all rhodamine spirolactam fluorescent probes employ the spiro ring-opening reaction either through metal ion coordination or metal ion-catalysed desulphurisation reaction. Having utilised a Hg²⁺-triggered domino reaction, Gong and his co-workers described the synthesis of a novel rhodamine thiospirolactam derivative, Hg-14 (Fig. 15), as an ‘off–on’ fluorescent probe for the detection of Hg²⁺ in neutral H₂O-MeOH (80/20; v/v) solution (Gong et al. 2012). Compound Hg-14 showed stability of response for Hg²⁺ sensing from 1.0 × 10⁸–1.0 × 10⁶ M. The fairly stable pH range of 3.0–11.0 was chosen for the conduct of the experiments. The fluorescence emission intensity of the rhodamine derivative Hg-14 underwent a marked increment (of about a 580-fold fluorescence improvement) upon the addition of 1 mM Hg²⁺. Contrastingly, the addition of 1 mM of Ag⁺ and 50 mM of the coexisting cations left no noteworthy influence on the probe’s fluorescence intensity. Whilst Probe Hg-14 showed linearity of detection response in the range of 10 nM–1 mM and an ultralow detection limit of 3 nM, its response time towards Hg²⁺ was not up to 6 min. As much as the opposite might have been desired, the reported compound Hg-14 was irreversible in its sensing nature towards Hg²⁺ (i.e. it was a chemodosimeter but not a probe), although it could bioimage Hg²⁺ in living HeLa cells with great sensitivity.

Hu and his teammates developed a simple yet effectual novel fluorescent compound, Hg-15 (Fig. 15), that anchors the thiorhodamine 6G-amide moiety by the employment of Hg²⁺-induced desulphurisation reaction and spirolactam opening of rhodamine (Hu et al. 2013). The compound, which functions as a chemodosimeter rather than as a probe, could detect Hg²⁺ in the broad and stable pH range of 6.0–10.0, although with the optimums pH range being 7.2–7.6. In 0.01 M HEPES-buffer ethanol solution (EtOH/H₂O 1:8, v/v, pH = 7.4) solution, the proposed compound Hg-15 revealed a remarkable increase in its fluorescence intensity only upon contact with Hg²⁺ in the co-presence of several other cations, which induced neither an individual nor a collective effect(s) on the fluorescence intensity of Hg-15. 5-min response time for the monitoring of Hg²⁺ by Hg-15 was observed. The Job’s method of continuous variation gives a 1:1 stoichiometric ratio between Hg-15 and Hg²⁺ in the Hg-15/Hg²⁺ complex. An estimated detection limit of 4.2 × 10^–8 M was obtained, and Hg-15 was successfully demonstrated as capable of monitoring Hg²⁺ in living cells via confocal laser scanning microscopy (CSLM).

Based on the spirolactam ring-opening process, Kumar et al. documented the rhodamine B phenyl hydrazide probe, Hg-16 (Fig. 16), being developed as a novel colourimetric and fluorescent chemodosimeter, for the sensitive, selective, and irreversible Hg²⁺ monitoring (Kumar et al. 2014). The fluorescence emission intensity was quite stable in the pH range of 7–12 upon the addition of various anions and cations. The compound which has the capability for naked-eye detection (from colourless to pink) of Hg²⁺ also displayed a substantial fluorescence increase in its emission intensity. At 580 nm, there was a significant strengthening of the fluorescence emission intensity of Hg-16 upon the addition of Hg²⁺ amidst several other cations and anions like Ca²⁺, K⁺, Mg²⁺, Pb²⁺, Ni²⁺, Zn²⁺, Fe³⁺, Cu²⁺, Cl⁻, NO₂⁻, NO₃⁻, S²⁻, SO₄²⁻, and ClO⁻ analysed. The linear detection response lied within the range 1–100 nM, with a detection limit of 0.019 nM calculated. The monitoring of Hg²⁺ by the probe was complete after about 1.5 min of the start of the reaction. The efficacious amphiphilic and fluorescence qualities of the designated compound Hg-16 were taken advantage of to bioimage Hg²⁺ in living MCF-7 cells by fluorescence microscopic studies. Table 1 summarises the analytical properties of Probe Hg-16 for Hg²⁺ detection.

Fig. 16

Structures of Probe Hg-16 and Chemodosimeter Hg-17 (Kumar et al. 2014; Wang et al. 2012)

Table 1 Reported analytical properties of Probe Hg-16 (Kumar et al. 2014)

Wang and colleagues devised a rhodamine-based compound, Hg-17 (Fig. 16), that anchors the thiospirolactam and benzimidazole moieties (Wang et al. 2012). The probe, which utilises the spirolactam ring-opening and desulphurisation process, functioned as a highly sensitive and selective ‘naked-eye’ chemodosimeter for Hg²⁺ sensing. In CH₃CN-HEPES buffer (0.01 M, pH 7.4) (3:7, v/v), the fluorescence intensity of the reported compound underwent a sharp increase in the order of 180-fold enlargement when Hg²⁺ was added to the probe solution, which was attributed to the selective spirolactam ring-opening process followed by desulphurisation. The other added cations like Zn²⁺, Pb²⁺, Cd²⁺, Co²⁺, Hg²⁺, Cu²⁺, Fe²⁺, Ag⁺, Al³⁺ and Cr³⁺ Zn²⁺, Pb²⁺, Cu²⁺, Fe²⁺, Ag⁺, Li⁺, Mn²⁺, Na⁺, Fe³⁺, Al³⁺, and Sr²⁺ induced no influence on the probe’s fluorescence emission intensity. The estimated analytical detection limit was found to be 7.4 nM.

Utilising a rosamine scaffold, Taki and co-workers gave an account of the purportedly strongest reversible fluorescent probe compound, Hg-18 (Fig. 17), designed for Hg²⁺ sensing (Taki et al. 2012). In 50 mM HEPES (pH 7.20, 0.1 M KNO₃) and DMSO (4:1, v/v), Hg-18 showed a stringent attraction towards Hg²⁺ amidst other foreign cations like Ni²⁺, Mn²⁺, Fe³⁺, Pb²⁺, Cu²⁺, Zn²⁺, Cd²⁺, and Co²⁺ with an ensuant 20-fold fluorescence increment observed. Cu⁺ and Ag⁺ interfered in the sensing of Hg²⁺ by Hg-18, although this was quite minuscule. The existence of a 1:1 stoichiometry between the probe and Hg²⁺ was demonstrated by the analysis of the result of the Job’s plot. The dissociation constant of 1.04 ± 0.05 × 10^–16 M was calculated for the generation of the complex between the said probe compound and Hg²⁺ analyte ion. The imaging ability of Hg-18 for Hg²⁺ HeLa cells was feasible.

Fig. 17

Structure of Probe Hg-18 (Taki et al. 2012)

Small-molecule Hg ²⁺ probes based on coumarin fluorophore

The coumarins, or benzo-α-pyrones, constitute a huge and important class of organic compounds. The structure of the simplest coumarin is made up of fused pyrone and benzene rings, with their position 2 harbouring a pyrone carbonyl group. Coumarins are naturally abundant, with the first coumarin isolated in 1820 from a bean variety, and with several other coumarin derivatives isolated from diverse plant species. The coumarins family exhibits exciting fluorescence properties, including high sensitivity to their local environment—in terms of both polarity and viscosity. This, in turn, led to their extensive application range desirably as sensitive fluorescent probes in a wide range of systems (Wagner 2009; Reddie et al. 2012).

Since the successful synthesis of the first thiocoumarin-derivative fluorescent probe for Hg²⁺ detection which exploits the desulphurisation of thiocoumarin to coumarin (Choi et al. 2009), there has been an upsurge in the subsequent synthesis of more such probes. Drawing inspiration from this, Qin and co-workers (Qin et al. 2018) disclosed a new ICT-based colourimetric and ratiometric fluorescent chemodosimeter, Hg-19 (Fig. 18), for Hg²⁺ monitoring. When excited at 477 nm and 543 nm, the probe is selective and sensitive towards Hg²⁺ detection in PBS buffer solution (10 mM, pH 7.4, containing 1% DMSO) in the coexistence of several other analytically relevant cations investigated (Na⁺, K⁺, Ca²⁺, Mg²⁺, Zn²⁺, Co²⁺, Cr³⁺, Cu²⁺, Fe²⁺, Fe³⁺, Ni²⁺, Pb²⁺, and Sn⁴⁺) . The probe’s analytical detection range was between 1.0 and 15.0 with a low detection limit of 1.85 ppb. Furthermore, the probe was successfully used for the fluorescence imaging of Hg²⁺ in live cells. The in vitro and in vivo application of the probe towards HeLa cells was ascertained.

Fig. 18

Structure of Hg-19 (Qin et al. 2018)

Based on Hg²⁺-induced cyclisation reaction, a simple 7-hydroxycoumarin-based compound, Hg-20 (Fig. 19), which acts as a ‘turn-on’ probe for the sensing of Hg²⁺ in aqueous media (CH₃CN/H₂O = 9:1, pH 7.0), was developed by Gao’s laboratory members (Gao et al. 2012). The probe is most effective in its sensing nature between the pH value of 6.2 and 8.0, whilst it could also maintain enough stability in this range (Fig. 19). Results revealed that there was a tremendous improvement in the compound’s fluorescence intensity upon the addition of Hg²⁺. However, in the absence of the competing metal ions, there was an insignificant variation in Hg-20′s fluorescence intensity, except for the quenching effect displayed by Cu²⁺. The results of the competition experiments further justified the high sensitivity and selectivity of Hg-20 towards Hg²⁺. The presence of anions also impacted a little effect on the fluorescence response behaviour of Hg-20 towards Hg²⁺. Probe Hg-20′s detection limit for Hg²⁺ monitoring was calculated to be 5.12 × 10^–7 M. The practical usage of the developed compound was demonstrated by its ability to measure Hg²⁺ in tap and well water samples.

Fig. 19

Structure of Probe Hg-20 (left), and the changes in the fluorescence of Probe Hg-20 (black line) and Probe Hg-20 with Hg²⁺(red line) in buffer solution as a function of increasing pH value (10 μM CH₃CN/H₂O = 9/1, v/v, excitation wavelength = 328 nm, and emission wavelength = 400 nm) (right) (Gao et al. 2012)

Five structurally similar coumarin-based fluorescent probes, Hg-21–Hg-25 (Fig. 20), that could selectively and sensitively monitor Hg²⁺ were developed (Guo et al. 2015). The authors reported that Hg²⁺ detection proceeded through the Hg²⁺-promoted desulphurisation of the probes—a seldom-used method for the construction of fluorescent probes. All the five probes (especially Probe Hg-21) possess excellent selectivity and anti-interference properties towards Hg²⁺ from other co-existing cations present together in the aqueous phase (Fig. 21). The probes showed linearity in their detection mode towards Hg²⁺ over the wide concentration range of 0.06–1.50 mM (0.06–0.90 mM for Probe Hg-25). The probes have interesting biological properties towards Hg²⁺, and the authors tested the efficacies of the probes (especially Probe Hg-21 because of its superb sensitivity for Hg²⁺ unmatched by the other probes) on MCF-7 cells. The in vitro cytotoxicity experiment revealed that Probe Hg-21 had little cytotoxicity on MCF-7 cells’ proliferation within the 0–10 mM concentration range. The binding mechanism was purported to have occurred via the Hg²⁺-promoted hydrolysis desulphurisation.

Fig. 20

Structures of Probes Hg-21–Hg-25 (Guo et al. 2015)

Fig. 21

Visual fluorescence emission of Probe Hg-21 (1 mM) in the presence of several cations (200 mM, 2.0 mM for Hg²⁺) on excitation at 365 nm using a UV lamp at ambient temperature (Guo et al. 2015)

The design of a water-soluble probe, Hg-26 (Fig. 22), which bears a coumarin unit as the fluorophore and a vinyl ether group as the receptor was documented by Wu’s lab (Wu et al. 2017). When investigated in HEPES buffer (20 mM, pH 7.0), there was a remarkable magnification of the probe’s fluorescence intensity upon the addition of Hg²⁺. The low detection limit (0.12 M) of Hg-26 proves that the probe exhibits outstanding selectivity and sensitivity towards Hg²⁺. The sensing mechanism of the probe with Hg²⁺ was confirmed using proton NMR (Fig. 22). The practical application of the probe as an excellent monitoring tool was demonstrated by its ability to detect traces of Hg²⁺ in real water samples (Table 2).

Fig. 22

The partial ¹H NMR (400 M, d₆-DMSO) spectra of Probe Hg-26 (I), Probe Hg-26 with Hg²⁺ after 1 h (II), and 7-hydroxy-4-methylcoumarin (III) (Wu et al. 2017)

Table 2 Monitoring of Hg²⁺ by Probe Hg-26 (Wu et al. 2017)

Small-molecule Hg ²⁺ probes based on naphthalimide and rhodamine hybrid fluorophore

Employing rhodamine 6G and rhodamine B appended to 4-Bromo-1,8-naphthalic anhydride as the fluorophore whilst using ethylenediamine as the spacer, Fang and co-workers successfully developed Probes Hg-27 and Hg-28 (Fig. 23) (Fang et al. 2015). Such systems have been proven effective for the highly sensitive and selective sensing of Hg²⁺ in the joint existence of other metal ions when spectroscopically investigated in CH₃CN/HEPES buffer (1:1, v/v, pH = 7.0). There was a 7.09-fold fluorescence enhancement when Hg²⁺ was added to a solution of Probe Hg-27 (Fig. 24), this being attributed to a Hg²⁺-chelated spirolactam ring opening. Meanwhile, the association constant and detection limit of Probe Hg-27 for Hg²⁺ monitoring are 8.31 × 10⁵ M⁻¹ and 7.91 × 10^–7 mol L⁻¹, respectively. The spectroscopic investigation of Probe Hg-28, a more water-soluble probe than Probe Hg-27, gave the association constant and detection limit of the binding of Hg-28 with Hg²⁺ as 4.36 × 10⁵ M⁻¹ and 5.46 × 10^–6 mol L⁻¹, respectively.

Fig. 23

Structures of Probes Hg-27 and Hg-28 (Fang et al. 2015)

Fig. 24

a Fluorescence titration spectra of Hg-28 (5 μM) in CH₃CN/HEPES buffer (1:1, v/v, pH = 7.0) in the presence of different concentrations of Hg²⁺ (0–16 μM). Inset: the plot of fluorescence intensity at 525 nm versus [Hg²⁺]. The excitation wavelength was 450 nm. b Fluorescence titration spectra of Hg-28 (5 μM) in pure CH₃CN in the presence of different concentrations of Hg²⁺ (0–24 μM). Inset: the image of fluorescence changes of Hg-28 (5 μM) before and after upon interaction with Hg²⁺ (24 μM) (Fang et al. 2015)

Xu’s laboratory synthesised Probe Hg-29 (Fig. 25), which was employed for the ratiometric detection of Hg²⁺ based on the FRET mechanism (Xu et al. 2017). Robust association constant of 2.75 × 10⁵ M⁻¹ was calculated, and minuscule detection limit of 0.059 μM was determined in EtOH/HEPES (v/v, 9:1, pH 7.0) in the analytical presence of other analyte ions, viz K⁺, Mg²⁺, Mn²⁺, Fe²⁺, Ni²⁺, Ba²⁺, Co²⁺, Na⁺, Zn²⁺, Cu²⁺, Pb²⁺, Ca²⁺, Cd²⁺, Ag⁺, Cr³⁺, and Hg²⁺. Thanks to its high recovery rate, real-time response, and anti-interference property, the probe was reportedly capable of being used for the ‘naked-eye’ Hg²⁺ monitoring and was also feasibly employed for Hg²⁺ monitoring in actual environmental water samples.

Fig. 25

Structure of Probe Hg-29 (Xu et al. 2017)

Small-molecule Hg ²⁺ probes based on rhodamine and coumarin hybrid fluorophore

Utilising the multiple advantages of ratiometric FRET process as a sensitive, selective, and adaptable detection technique, Zhou et al. (2013) came up with a Hg²⁺-selective probe, Hg-30 (Fig. 26), built from the rhodamine–coumarin scaffold. This probe was designed to exhibit a dual-responsive fluorescent and colourimetric analytical detection (Fig. 26) when investigated for Hg²⁺ selectivity when jointly present with common cations like Fe²⁺, Mn²⁺, Ni²⁺, Co²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Pb²⁺, and Cr³⁺ in CH₃CN/H₂O system. Upon the addition of these metal ions, there was no noticeable change in the probe’s emission intensity. The designated probe was capable of the biological imaging of intracellular Hg²⁺.

Fig. 26

Absorption (up) and fluorescent (down) spectra of Probe Hg-30 (2 μM) (inset) (HEPES/CH₃CN, v/v = 20:80; pH 7.0) (Zhou et al. 2013)

Small-molecule Hg ²⁺ probes based on naphthalimide and coumarin hybrid fluorophore

Through a mercury-promoted desulphurisation of thiocarbonyl moiety, a FRET dyad, Hg-31, which bears both 1,8-naphthalimide and rhodamine B moieties, was constructed as a ratiometric fluorescent probe for Hg²⁺ detection (Liu et al. 2012). Compound Hg-31 (Fig. 27) is most appropriate for sensing application within the pH range of 5.7 to 11.0. Meanwhile, the linear range of fluorescence response detection was 4.2–10 mM, and the response time for the sensing investigation was 10 min. In (2:1, v/v) MeOH/H₂O solution (10 mM Tris–HCl, pH 7.0), there was a monumental fluorescence intensity increment in Hg-31 explained as due to the rhodamine B unit ring opening. Cations like K⁺, Na⁺, Fe²⁺, Mg²⁺, Cr³⁺, Fe³⁺, Ca²⁺, Zn²⁺, Co²⁺, Cu²⁺, Pb²⁺, Ni²⁺, Cd²⁺, Ag⁺, Au³⁺, and Pb² impacted zero changes on the fluorescence intensity of Compound Hg-31, albeit there was a little interference induced by Ag⁺.

Fig. 27

Structure of Probe Hg-31 (Liu et al. 2012)

Small-molecule Hg ²⁺ probes based on rhodamine and coumarin hybrid fluorophore

Through a spirocyclisation-modulated process, a rhodamine receptor was fused with a 7-diethylaminocoumarin fluorophore to develop a NIR ratiometric fluorescent Probe Hg-32 (Fig. 28) (Liu et al. 2013a). The designed compound was stable in the long pH range of 1 to 12: When Hg²⁺ was increasingly added to the solution of the probe in Tris–HCl/CH₃CN (10 mM, pH = 7.4, 1:1, v/v) amidst cations like, K⁺, Na⁺, Mg²⁺, Cu²⁺, Ca²⁺, Fe²⁺, Zn²⁺, Fe³⁺, Ni²⁺, Cr²⁺, Mn²⁺, Cd²⁺, Pb²⁺, Ag⁺, and Co²⁺, there was a massive downward shift in the fluorescence intensity at 480 nm, only for a new emission band positioned at 695 nm to appear. Desirably, the sensing reaction showed completion only after about 10 s of the starting time. A commendable linear ratiometric response (I₆₉₅/I₄₈₀) of Hg²⁺ monitoring was observed in the concentration range of 0–30 mM with a detection limit of 2.8 × 10^–8 M estimated. Probe Hg-32 could image Hg²⁺ in living HeLa cells.

Fig. 28

Synthetic pathway to fluorescent Probe Hg-32 (Liu et al. 2013a)

Small-molecule Hg ²⁺ probes based on rhodamine and pentaquinone hybrid fluorophore

Based on a TBET paradigm, Bhalla et al. documented the first instance of pentaquinone derivatives, Hg-33 and Hg-34 (Fig. 29), joined with a rhodamine unit devised for the ultra-high selective and sensitive tracking of Hg²⁺ (Bhalla et al. 2012). The optimum working pH range of the compounds Hg-33 and Hg-34 is 4.0–7.0. In THF/H₂O (9.5:0.5, v/v), only Hg²⁺ led to the enhancement of the fluorescence intensities of Hg-33 and Hg-34 (56-fold and 160-fold increment, respectively) in the midst of the other co-existent metal ions. The sensing performances of Hg-33 and Hg-34 were not affected by counter anions, except for NO₃⁻ that left a slight decrement in the sensing property of receptor Hg-34. The method of continuous variation, i.e. Job’s plot, furnishes 1:1 and 1:2 stoichiometric ratios of Hg²⁺ interaction with receptors Hg-33 and Hg-34, respectively. The calculated detection limits of receptors Hg-33 and Hg-34 for Hg²⁺ monitoring are 5 × 10^–6 and 7 × 10^–7 M, respectively. The receptors Hg-33 and Hg-34 displayed reversibility in their Hg²⁺ abilities. The receptor Hg-34 was successfully appraised for in vitro Hg²⁺ monitoring in prostate cancer (PC3) cell lines.

Fig. 29

Structures of Probes Hg-33 and Hg-34 (Bhalla et al. 2012)

Small-molecule Hg ²⁺ probes based on boron-dipyrromethene (BODIPY) fluorophore

The 4,4-difluoro-4-bora-3a,4a-diazas-indacene molecule whose structure can be easily tuned at the carbon positions 1,3,5,7, and 8 is termed boron-dipyrromethene (BODIPY) chromophores, the BODIPY itself being registered under the trademark of Molecular Probes Inc. (Keller et al. 1995; Schade et al. 1996; Jones et al. 1997; Durand et al. 2000; Marme et al. 2003; Traverso et al. 2003). Treibs and Kreuzer 1968 reported the first BODIPY chromophore. The useful photophysical characteristics of BODIPY-based probes have led to their increasing usage. There is operational ease in the tuning of their visible excitation and emission by simply modifying the pyrrole core (Loudet and Burgess 2007; Thoresen et al. 1998; Rurack et al. 2001). They also possess high fluorescence quantum yields (Φ_F ≈ 1.0, even in water), high molar absorption coefficients (typically log max > 8.8), narrow emission bandwidths, and excellent photostability. They lack a net ionic charge, and their fluorescence is insensitive to solvent or pH. All these desirable properties constitute additional advantages (Sathyamoorthi et al. 1994; Treibs and Kreuzer 1968; Haugland et al. 1988).

Maity’s lab documented a fluorescent probe for Hg²⁺, Hg-35 (Fig. 30), which incorporates a 2,2′-(ethane-1,2-diylbis(oxy))bis(N,N-bis(pyridine-2-ylmethyl)aniline receptor moiety built from a BODIPY derivative (Maity et al. 2015). Hg-35 exhibited a large variation in its fluorescence within the pH range 4–8. The compound showed both selectivity and sensitivity towards Hg²⁺ (or Cd²⁺) when present with an array of other cations such as Li⁺, K⁺, Na⁺, Mn²⁺, Mg²⁺, Co²⁺, Ca²⁺, Fe²⁺, Ni²⁺, Zn²⁺, Cu²⁺, and Ag⁺ in 20% DMSO-H₂O medium. The compliance of the selectivity of the compound for Hg²⁺ was justified by the great fluorescence enhancement noticed upon the addition of Hg²⁺. The analysis of the Job’s plot yielded a 1:1 binding stoichiometry of the Hg-35/Hg²⁺ complex. The estimated value of the association constant is 1.8 × 10⁵ M⁻¹ for Hg²⁺ (and 3.77 × 10⁴ M⁻¹ for Cd²⁺), and a detection limit of 38 nM was obtained. Compound Hg-35 detected Hg²⁺ (or Cd²⁺) well enough in living cells.

Fig. 30

Structure of Probe Hg-35 (Maity et al. 2015)

The BODIPY derivative, Hg-36 (Fig. 31), is a fluorescence probe developed for the highly sensitive and selective monitoring of Hg²⁺ in CH₃OH (Vedamalai 2012). An array of cations (Ag⁺, K⁺, Cd²⁺, Ca²⁺, Co²⁺, Fe²⁺, Cu²⁺, Fe³⁺, Mg²⁺, Mn²⁺, Ni²⁺, Zn²⁺, and Pb²⁺) was used to assess the interference property of the compound in the detection of Hg²⁺. Desirably, these cations impacted only a meagre effect on Hg-36′s fluorescence emission intensity. The apparent dissociation constant of Hg-36/Hg²⁺ was calculated to be 62 μM. The sensing property of Hg-36 was tested by mixing it with other cations like Ag⁺, Ca²⁺, Cd²⁺, Co²⁺, Cu²⁺, Fe²⁺, Fe³⁺, K⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pb²⁺, and Zn²⁺. Experimental results revealed that the fluorescence emission intensity of the BODIPY derivative underwent a substantial increase upon the addition of Hg²⁺, but remained unperturbed when other metal ions were added. Job’s method indicated a 1:1 binding ratio between the detecting compound and the detected ion. The dissociation constant of 62.1 ± 5.7 μM was obtained. Meanwhile, a detection limit of 2.8 μM was estimated. Utilising the fluorescence rationale, Hg-36 proves very suitable for Hg²⁺ tracking in living HeLa cells.

Fig. 31

Structure of Probe Hg-36 (Vedamalai 2012)

Small-molecule Hg ²⁺ probes based on cinnamaldehyde fluorophore

Kumar and co-workers synthesised a PET fluorescence ‘turn-on’ probe, Hg-37 (Fig. 32), that anchors N,N-dimethylaminocinnamaldehyde, for the monitoring of Hg²⁺, whereby the as-prepared complex Hg-37/Hg²⁺ was further engaged for the detection of picric acid (Kumar et al. 2012b). In THF/H₂O (9:1, v/v), recognition and competitive experiments revealed that the addition of metal ions to the probe’s solution did not induce any substantial change in the fluorescence intensity of the probe, except for Hg²⁺ that impacted a fluorescence quenching phenomenon. The experimental result provides a detection limit of 80 × 10^–9 M for Hg²⁺ tracking by the designed compound. Both the linear regression analysis (SPECFIT programme) and Job’s plot indicated a 1:2 stoichiometry of chelation between the probe and Hg²⁺ and a binding constant of 9.16 ± 0.03 was estimated. It was demonstrated that the interaction between Hg²⁺ and compound Hg-37 was reversible.

Fig. 32

Structure of Probe Hg-37 (Kumar et al. 2012b)

Small-molecule Hg ²⁺ probes based on Schiff base fluorophore

The Schiff base reaction was conceptualised in 1864 by the German chemist, Hugo Schiff, which is a reaction between aldehyde- or ketone-containing compounds and amino groups, leading to the generation of imine groups (–CN–) (Fig. 33). The set of reactions constitute popular chemical reactions due to that their reaction conditions are mild and that their reaction rates are high (Jia and Li2015). Only in the past few years had Schiff base compounds become increasingly applied for the generation of fluorescent and colourimetric probes.

Fig. 33

Schematic demonstration of Schiff base reaction (Jia and Li 2015)

Mandal et al. constructed a series of new imine-based probes, Hg-38, Hg-39, and Hg-40 (Fig. 34), for the sharp fluorescence ‘turn on’ detection of Hg²⁺ in CHCl₃/CH₃CN (1:4, v/v) over other competing metal ions (Mandal et al. 2012). There was a monumental increase in the fluorescence intensities of Hg-38, Hg-39, and Hg-40 upon interaction with Hg²⁺, this being explained as owing to imine isomerisation and inhibited PET mechanism. The composite binding constants of Hg²⁺ by Hg-38, Hg-39, and Hg-40 were obtained to be 2.79 × 10⁹ M⁻² L² (for Hg-38) and 1.29 × 10⁹ M⁻² L² (for Hg-39 and Hg-40). Meanwhile, the analysis of the Benesi–Hildebrand plots for Hg-39 and Hg-40 gave a 2:1 stoichiometric ratio. Hg-38 was reversible in its sensing of Hg²⁺ after the addition of KI that led to the recovery of the unbound probe compound from the Hg-38/(Hg²⁺)₂ complex. Interestingly, Hg-38 and Hg-39 were successfully demonstrated for Hg²⁺ detection in living cervical cancer HeLa cells.

Fig. 34

Structures of imine-based Probes Hg-38, Hg-39, and Hg-40 (Mandal et al. 2012)

Quang reported the synthesis of the ICT aminothiourea-derived Schiff base, Hg-41 (Fig. 35), for the fluorescent recognition of Hg²⁺ (Quang et al. 2013). In aqueous solution, the addition of Hg²⁺ to the probe’s solution pushed the fluorescence intensity upward at about 30% increment. Contrastingly, competitive cations like K⁺, Cd²⁺, Pb²⁺, Cr³⁺, Fe²⁺, Ni²⁺, Zn²⁺, Co³⁺, Ca²⁺, Mg²⁺, and Na⁺ led to benign variation in the fluorescence of Hg-41. The most desirable working pH range of Hg-41 for Hg²⁺ determination was from 5 to 9. Moreover, the time required for the reaction to be complete was 30 s. A 2:1 complex of Probe Hg-41 and Hg²⁺ was derived, which was confirmed by the result of the ESI–MS.

Fig. 35

Proposed interaction mechanism between Probe Hg-41 and Hg²⁺ (Quang et al. 2013)

The novel and elegant pyrene-based free thiol probe, Hg-42 (Fig. 36), that bears the Schiff base was documented by Shellaiah and co-workers (Shellaiah et al. 2015). The developed compound, which utilises a reversible chelation-enhanced fluorescence (CHEF) mechanism, was engaged as a fluorescence ‘turn on’ probe for the tracking of Hg²⁺ ion in DMSO/H₂O (v/v = 7/3; pH 7.0). The original, strong fluorescence of the devised probe was enhanced when Hg²⁺ was added to it, whereas other cations including Na⁺, Ag⁺, Ni²⁺, Co²⁺, Fe³⁺, Zn²⁺, Cd²⁺, Pb²⁺, Cr³⁺, Cu²⁺, Mg²⁺, Hg²⁺, Mn²⁺, Fe²⁺, and Al³⁺ did not impact a noticeable effect on the fluorescence intensity of the probe, in either single or dual metal analyses. The only exception was Pb²⁺ and Cd²⁺ that interfered somewhat weakly. Job’s plot analysis gives a distinct 2:1 binding ratio of the derived probe-Hg²⁺complex. The detection limit and association constant are 2.82 × 10^–6 M and 7.36 × 10⁴ M⁻¹, respectively. Desirably, the usage of Hg-42 for Hg²⁺ imaging in living HeLa cells was successfully established.

Fig. 36

Structural representation of Probe Hg-42 (Shellaiah et al. 2015)

Small-molecule Hg ²⁺ probes based on anthracene fluorophore

Srivastava and teammates synthesised an effective PET compound, Hg-43 (Fig. 37), which anchors anthracene and benzhydryl moieties via a piperazine bridge, as a fluorescent molecular probe for Hg²⁺ sensing (Srivastava et al. 2014). There was observed a large increase in the fluorescence intensity of ca. tenfold in HEPES buffer and ca. 15% in CH₃CN-H₂O solution, with a consequential colour change from blue to blue-green. Other cations (Na⁺, K⁺, Ca²⁺, Zn²⁺, Pb²⁺, Ag⁺, Cd²⁺, Co²⁺, Ni²⁺, Cu²⁺ Fe²⁺, Fe³⁺, Mg²⁺, Ca²⁺, and Zn²⁺) did not engender any noteworthy influence on the fluorescence intensity of Hg²⁺, except for Fe³⁺ that interfered quite slightly (ca. 5 to 7%) on Hg-43′s fluorescence emission intensity. The probe existed with Hg²⁺ in Hg-43/Hg²⁺ complex in a 1:2 stoichiometry. Hg-43 was found to be pH insensitive within the range 7–14, with pH 5 being the optimal pH value. Job’s plot analysis revealed a 1:2 stoichiometric binding interaction between Hg-43 and Hg²⁺. The association constant and detection limit were obtained as 1.06 × 10¹⁰ M⁻² and 10 nM or 2 ppb, respectively. The probe Hg-43 was successfully applied as a solid probe for Hg²⁺ monitoring on solid surfaces like paper strips and slides coated with silica, as well as to bioimage Hg²⁺ in HeLa cells.

Fig. 37

Structure of Probe Hg-43 (Srivastava et al. 2014)

Praveen and co-workers constructed the anthracene-oxyquinoline dyad, Hg-44 (Fig. 38), for Hg²⁺ sensitive and selective sensing (Praveen et al. 2012). In MeCN-H₂O system, Hg²⁺ addition quenched the fluorescence of the reported compound, a phenomenon unobserved in the cases of other co-existed cations. A 1:1 binding stoichiometry was provided by the analysis of the Job’s plot, whilst a detection limit of 3.2 × 10^–6 M was calculated from the Benesi–Hildebrand plot. Probe Hg-44 was reversible in its tracking of Hg²⁺.

Fig. 38

Plausible binding mode of Probe Hg-44 with Hg²⁺ (Praveen et al. 2012)

 A new anthracene-based sensing modality, Hg-45, 1-(Anthracen-2-yliminomethyl)-napthalen-2-ol (AIN) (Fig. 39), which features aggregation-induced emission (AIE) was recently disclosed by Shyamal’s group (Shyamal et al. 2018). The probe exhibited a suppression in its fluorescence intensity upon selective recognition of Hg²⁺ with a linearity of response within the concentration range 0.3–3.6 M. The sensing ability of the probe was tested in THF/H₂O (7:3, v/v) in the presence of various relevant metal ions. The probe’s sensitivity was excellent (a limit of detection limit of ca. 3 ppb was estimated), and the quenching constant obtained from Stern–Volmer equation was 2.54 × 10⁵ M⁻¹. The probe’s ability to quantify Hg²⁺ concentration in real water samples was successfully investigated.

Fig. 39

Structure of Probe Hg-45, 1-(Anthracen-2-yliminomethyl)-napthalen-2-ol (AIN) (Shyamal et al. 2018)

Small-molecule Hg ²⁺ probes based on squaraine fluorophore

The squaraines are a subclass of the polymethine dyes possessing a zwitterionic structure and constituting one of the most fascinating groups of dyes for the preparation of near-infrared fluorescent probes (Oswald et al. 2000; Kukrer and Akkaya 1999; Dilek and Akkaya 2000). They exhibit outstanding physicochemical properties, including magnificent and sharp absorption bands, (detectable in the visible and NIR regions) as well as intense fluorescence bands (Welder et al. 2003; Kim et al. 2002). These properties make the squarylium fluorophores suitable for sensing purposes. Infusion of substituents into the aromatic ring or on the N atom of the terminal heterocyclic moiety could lead to their structural modifications. Such variations can be used to produce a red shift of the absorption and fluorescence bands.

Fan and teammates developed the squaraine-appended colourimetric and fluorescent probe, Hg-46 (Fig. 40) (Fan et al. 2012), for Hg²⁺-sensitive and selective sensing. In ethanol–phosphate buffer (EtOH/PB) (50:50, v/v) solution, Hg²⁺ addition amidst several other metal ions led to a gradual dampening of the probe’s fluorescence intensity. Job’s plot analysis indicates the formation of a 1:1 binding stoichiometry. The calculated association constant and detection limit are 3.05 × 10³ M⁻¹ and 1.3 × 10^–7 M, respectively. Experimental outcomes demonstrated Hg-46 as reversible in its sensing mode, i.e. the unbound Hg-46 was regenerated upon EDTA addition to Hg-46/metal complex’s solution.

Fig. 40

Structure of the squaraine Probe Hg-46 (Fan et al. 2012)

Small-molecule Hg ²⁺ probes based on pyrene fluorophore

Weng and laboratory members reported a compound, Hg-47 (Fig. 41), that anchors the pyrene skeleton and diazene architecture (Weng et al. 2012). The designated compound acts as a useful fluorescent probe for tracking Hg²⁺. In CH₃CN solution, the addition of various cations did not incite any tremendous change in the fluorescence of Hg-47, whereas the addition of Hg²⁺ led to a marked decrement in the fluorescence of Hg-47, affirming the affinity of the designed probe for Hg²⁺ even in competition with other analytically investigated cations. A 2:1 stoichiometric ratio of the interaction of the probe and Hg²⁺ was furnished by Job’s plot analysis, which was corroborated by MS and NMR experimental results. The detection limit was calculated to be 4.69 × 10^–6 M.

Fig. 41

Possible binding mechanism of Probe Hg-47 with Hg²⁺ (Weng et al. 2012)

Small-molecule Hg ²⁺ probes based on rhodamine and pyrene hybrid fluorophore

Being informed by the knowledge that rhodamine and pyrene scaffolds exhibit excellent optical and configurable characteristics, Rui et al. developed two rhodamine–pyrene conjugated probes, Hg-48 and Hg-49 (Fig. 42) (Rui et al. 2016), which display strong affinities to Hg²⁺ via FRET process in the existence of other cations tested in EtOH/H₂O (1:1, v/v, pH 7.0). The detection limit of sensing of Hg²⁺ by Probes Hg-48 and Hg-49 is 4.34 × 10^–7 mol L⁻¹ and 1.91 × 10^–5 mol L⁻¹, respectively. The experimental results showed that Probe Hg-48 is a better probe than Probe Hg-49 due to its shorter response time, lower detection limit, and reversibility. Moreover, Probe Hg-48 was effectively employed for Hg²⁺ tracking in real water samples.

Fig. 42

Structures of Probes Hg-48 and Hg-49 (Rui et al. 2016)

Two rhodamine–pyrene probes, Probes Hg-50 and Hg-51 (Fig. 43), were synthesised by Chu’s laboratory (Chu et al. 2013). In EtOH/H₂O (2:1, v/v), both these probes exhibited outstanding sensing properties towards Hg²⁺ when tested in the analytical presence of commonly tested cations. The limit of detection of Probe Hg-50 for Hg²⁺ monitoring was quantified as 0.72 μM, whilst that of Probe Hg-51 was obtained as 5.3 μM. Meanwhile, the association constants of these two probes for Hg²⁺ monitoring were 2.74 × 10⁵ M⁻¹ and 1.56 × 10⁵ M⁻¹, respectively. It was experimentally confirmed that Probe Hg-50 had better spectral properties than Probe Hg-51, in terms of higher selectivity, excellent reversibility, and lower detection limit than Probe Hg-51, due to the thiophilic property of Hg²⁺. Finally, the utility of Probe Hg-50 for Hg²⁺ detection in practical biological systems was successfully demonstrated.

Fig. 43

Structures of Probes Hg-50 and Hg-51 (Chu et al. 2013)

Small-molecule Hg ²⁺ probes based on azo derivative fluorophore

Tian et al. documented an azo derivative, Hg-52 (Fig. 44), based on alkynes, which served as a colourimetric and fluorescence probe for the detection of Hg²⁺ (Tian et al. 2014). The probe gave no fluorescence emission in aqueous solution, which is atypical of the azo group of probes. But the addition of Hg²⁺ amongst several other cations and anions led to a 168-fold increase in the probe’s fluorescence intensity. The Job’s plot provided a 1:1 stoichiometric ratio in the interaction of Hg-52 with Hg²⁺. The detection limit and association constant of the probe for the sensing of Hg²⁺ were calculated as 46.50 nM and 3.96 × 10⁶ M¹, respectively. The sensing mechanism of Hg²⁺ by Hg-52 was demonstrated reversible by the addition of Na₂S. The specificity of Probe Hg-52 towards Hg²⁺ was determined by fluorescence screening. Hg-52′s potency to track Hg²⁺ was successfully demonstrated in living HT-29 cells.

Fig. 44

Structures of Probes Hg-52 and Hg-53 (Tian et al. 2014; Yu et al. 2016)

Yu and co-workers designed a thioacetal probe, Hg-53 (Fig. 44), that anchors both carbazole and azobenzene units (Yu et al. 2016). The spectroscopic characterisation of the compound showed that it functioned effectively in the range of 0–2.5 M and possessed a short response time of 30 s. In CH₃CN/H₂O solution, various cations (including Na⁺, K⁺, Ca²⁺, Mg²⁺, Ni²⁺, Li⁺, Co²⁺, Ag⁺, Cu²⁺, Pb²⁺, Mn²⁺, Cr³⁺, Fe³⁺, Zn²⁺, Cd²⁺, Sr²⁺) impinged no monumental change in Hg-53′s fluorescence intensity except for Hg²⁺ that led to a dramatic increment in the fluorescence intensity of the designated compound (about a 24-fold increase). Job’s plot indicates a 1:1 binding stoichiometry between the probe Hg-53 and Hg²⁺. A detection limit of 1.80 × 10^–8 M was calculated.

Small-molecule Hg ²⁺ probes based on benzimidazole derivative fluorophore

Hu et al. (2015) reported on a novel Hg²⁺ non-sulphur fluorescent probe, Hg-54 (Fig. 45) that incorporates the benzimidazole and quinoline moieties as the fluorescence signal groups. The compound exhibited a sharp distinguishing ability for Hg²⁺ over an array of other divalent cations as monitored by fluorescence spectroscopic analysis in H₂O/DMSO (1:9, v/v) solution with a massive colour change from blue to colourless observed when Hg²⁺ was added to the probe’s solution in aqueous media. Similar experiments for competitive cations present did not induce any prominent variation in the colour. The analytical limit of detection of the fluorescence response of the probe to Hg²⁺ is low down to 9.56 × 10^–9 M. The usage of the probe in the fabrication of test kits which could monitor Hg²⁺ demonstrated the probe’s practical application utility in an aqueous environment.

Fig. 45

Synthetic route to Probe Hg-54 (Hu et al. 2015)

Small-molecule Hg ²⁺ probes based on the phenanthroline fluorophore

Shan and co-workers developed a fast, responsive, and highly selective Hg²⁺ probe, Hg-55 (Fig. 46), based on 2,9-dimethyl-1,10-phenanthroline (2,9-DMP) fluorophore (Shan et al. 2016). The selectivity and sensitivity experiments were carried out in Tris-HNO₃ at the optimum sensing pH value of 2.0. The probe’s response to Hg²⁺ was rapid and selective within a good linear range of 0.05–2.00 µM. The reported compound was then utilised to monitor concentrations of Hg²⁺ in aqueous samples of drinking water and Zhujiang River water. Excellent recoveries of 85.42–101.5% and 81.72–96.09%, respectively, were obtained for each of these samples. Fluorescence titration experiment gave a 1:1 complex, demonstrating a 1:1 stoichiometric mode between the probe and Hg²⁺. This was then further evidenced by the results of Job’s plot and single-crystal X-ray diffraction.

Fig. 46

Structure of 2,9-DMP fluorophore-based Probe Hg-55 (Shan et al. 2016)

Small-molecule Hg ²⁺ probes based on miscellaneous fluorophores

Because of limited space, it is difficult to cover all fluorophores used in the development of small-molecule Hg²⁺ probes in this review work. However, apart from the small-molecule Hg²⁺ probes highlighted above, other fluorophores such as cyanine and fluorescein (Fig. 47) (Zheng et al. 2013; Ando and Koide 2011; Liu et al. 2013b) have been employed as alternative receptors in design and synthesis of small-molecule Hg²⁺ probes. Zheng et al. (2013) provided an overview of fluorescent probes based on the xanthene scaffolds, predominantly the rhodamine and fluorescein moieties. The fluorescent behaviours of these probes towards diverse analytes, including mercuric ion (Hg²⁺) or methylmercury species (MeHg), were discussed. Ando and Koide (2011) developed a new vinyl ether Hg-selective probe by the reaction of an allyl ether and KO^tBu in DMSO through the oxymercuration reaction. The probe, which functions best at pH 4 in 50 mM phthalate buffer, detects Hg²⁺ at ambient temperature (i.e. 25 °C) and can be applied for Hg²⁺ monitoring in river water and dental amalgam samples. A cyanine-modified nanoprobe (the cyanine dye was assembled on lanthanide UCNPs’ surface) which functions as a dual colourimetric and fluorescent probe for MeHg⁺ sensing was reported by Liu’s laboratory (2013b). This probe, whose sensing properties were largely uninfluenced by the presence of other non-mercuric cations, gave the detection limit of ca. 200 ppb. The probe showed great potential imaging applications in living cells and also for luminescence in vivo imaging.

Fig. 47

Simplest structural representations of cyanine and fluorescein moieties (Zheng et al. 2013; Andoand Koide 2011; Liu et al. 2013b)

The morpholine-based fluorophore possesses a lysosome-targeting capability and has been found efficient as a lysosomal localisation group. Informed by this, Zhang’s group developed a PET dual-functional colourimetric and ratiometric fluorescent probe, Hg-56 (Fig. 48), from the 4-chloro-7-nitrobenzo-2,1,3-oxadiazole (NBD-Cl) moiety (Zhang et al. 2016). The probe is water-soluble, bio-compatible and cell permeable, and could, therefore, monitor Hg²⁺-related cellular toxicological actions of HeLa cells.

Fig. 48

Structure of Probe Hg-56 (Zhang et al. 2016)

Recently, Gu et al. and Ding and co-workers synthesised and evaluated the optical features of probes based on the 2-(2′-hydroxyphenyl)-9,10-phenanthroimidazole (HPI) and dithioacetal fluorophores, Hg-57 and Hg-58 (Fig. 49), respectively (Gu et al. 2015; Ding et al. 2015). Both these probes have superior binding properties towards Hg²⁺ in the coexistence of other analytically investigated cations.

Fig. 49

Structures of Probes Hg-57 and Hg-58 (Gu et al. 2015; Ding et al. 2015)

The AIE feature of a π-conjugated cyanostilbene derivative of (Z)-2-(4-nitrophenyl)-3-(4-(vinyloxy)phenyl)acrylonitrile, Hg-59 (Fig. 50), was advantage taken of by Wang et al. in their design of a new sensing modality (Wang et al. 2015). The sensing properties of the designated probe compound were carried out in THF/H₂O (2:8, v/v). Probe Hg-59 was selective towards Hg²⁺ amidst an array of metal ions concomitantly investigated, and the fluorescence intensity of Hg-59 was dampened in a linear fashion with the incremental addition of Hg²⁺ (0–50 μM). The selectivity property of the probe was then tested with the probe’s detection limit calculated as 37 nM. These good properties of the probe enabled the quantification of Hg²⁺ in real water samples, thereby lending validity to the probe’s good sensing ability.

Fig. 50

Structure of Hg-59 (Wang et al. 2015)

Hg ²⁺ probes based on supramolecular fluorophores

 The seminal work of Chen and co-workers (2017) represented the first case of the use of supramolecular polymers in sequestering toxic heavy metal ions. Their supramolecular system was prepared through self-assembly of a thymine-substituted copillar[5]arene, 1, (catcher) and a tetraphenylethylene (TPE) derivative, 2 (indicator), together with Hg²⁺. The reversible recognition studies of  Hg²⁺ with the thymine moieties of 1 were investigated. The host–guest interaction between 1 and 2 was also examined, and the supramolecular complex/pseudorotaxane 2 ⊂ 1 was then used for Hg²⁺ tracking. Fluorescence titration experiments in the presence of other cations in 300 μM (CH₃)₂CO showed that there was a massive enhancement of the absorption maximum of 1 upon the incremental addition of 1.0 equiv. of Hg²⁺ coupled with a bathochromic shift from 326 to 329 nm. This was being accrued to the coordination of Hg²⁺ with the thymine moieties in 1, leading to the formation of T − Hg²⁺ − T pairings between 1 and Hg²⁺. TPE derivatives are known to only display strong fluorescence after aggregation; otherwise, they exhibit zero or low fluorescence when existent in a highly dispersed state. Springing from the AIE properties of 2, the derived supramolecular polymer exhibited strong fluorescence which was exploited for Hg²⁺ detection and removal. Linearity of fluorescence response in the range 0 − 180 μM was observed, and the analytical detection limit was 2.3 μM. The applicability of the 2 ⊂ 1 supramolecular system in removing Hg²⁺ from real water samples was successfully tested.

 Taking advantage of supramolecular self-assembly, a new sensitive and selective probe, (E)-1-((5-(4-nitrophenyl) furan-2-yl)methylene) semicarbazone (Hg-60) (Fig. 51), was derived from fusing 5-(4-nitrophenyl)-2-furan (the fluorophore) with semicarbazide groups (Qu et al. 2014). In DMSO/H₂O (8:2, v/v) HEPES buffer (pH = 7.2) solution, there was a dampening of the fluorescence intensity of the probe (20 μM) when Hg²⁺ (1 equiv.) was added in the presence of Fe³⁺, Hg²⁺, Ag⁺, Ca²⁺, Cu²⁺, Co²⁺, Ni²⁺, Cd²⁺, Pb²⁺, Zn²⁺, Cr³⁺, and Mg²⁺ (20 μM). This observation was ascribed to the supramolecular self-assembly breaking and the cooperation reaction occurrence. The detection limit of Hg-60 for Hg²⁺ determination was 2.084 nM, whilst Job’s plot analysis furnished a 1:2 stoichiometric ratio between Hg-60 and Hg²⁺ in Hg-60–Hg²⁺. Test strips of the probe successfully tracked Hg²⁺ in solution.

Fig. 51

Structure of Hg-60 (Qu et al. 2014)

Hg ²⁺ probes based on carbohydrate-appended resorufin fluorophore

Ma and co-workers, with the employment of a carbohydrate-based Ferrier carbocyclisation reaction, designed a water-soluble fluorescent probe, Hg-61 (Fig. 52), for the monitoring of Hg²⁺ through a ‘turn on’ mode of its fluorescence intensity (Ma et al. 2012). The probe’s stability was established within the wide pH range of 3.4–9.3; the best operational pH value of the designated probe was 7.4. Meanwhile, 15-min response time for the sensing of Hg²⁺ by the reported probe was achieved. The experimental pH range employed for the analytical experiments is 3.4–9.3. In pure H₂O (20 μM, pH 6.0), the compound maintained no fluorescent character. However, when the pH changed to > 6.96 by the addition of PBS buffer, there was a sudden appearance of vibrant fluorescence intensity with an accompanying colour variation from colourless to purple. This is due to the formation of O-anionic compound from the resorufin in the probe. The incremental additions of Hg²⁺ (0–1.0 μM) incited a 25-fold enhancement of the compound’s fluorescence intensity. However, the addition of a dozen of cations including Ca²⁺, Cd²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺, Zn²⁺, Ag²⁺, Pb²⁺, Fe³⁺, Fe²⁺, Ni²⁺ did not alter the fluorescence intensity significantly under the similar investigation conditions. The limit of detection of Probe Hg-61 for Hg²⁺ was quantified as 0.15 μM or 30 μg/L. The potent value of Compound Hg-61 was realised in its application to track Hg²⁺ analyte ion in the A549 cell line of a 5-day-old zebrafish and human lung adenocarcinoma epithelial origins (Fig. 53).

Fig. 52

Structure of Probe Hg-61 (Ma et al. 2012)

Fig. 53

Hg²⁺ fluorescence images in A549 cells and zebrafish with Probe Hg-61. Bright-field transmission image (AC) and fluorescence image (DF) of A549 cells pre-treated with 20, 20, and 100 μM of Probe Hg-61 for 24 h and then incubated with 0, 10, and 50 μM of Hg²⁺ for 60 min, respectively (under green light excitation). Zebrafish bright-field transmission image (G–I) and fluorescence image (J–L) pretreated with 30, 30, and 60 μM of Probe Hg-61 for 120 min followed by incubation with 0, 15, and 30 μM of Hg²⁺ for 30 min, respectively (under green light excitation) (Ma et al. 2012)

Small-molecule Hg ²⁺ probes based on polymer-embedded fluorophore

A novel water-soluble non-conjugated fluorescent polymer dots (PDs), G-PEI PDs, synthesised by reacting polyethylenimine (PEI) and glutathione (GSH) was disclosed from Luo’s laboratory in 2018 (2018). In Britton–Robinson buffer (0.04 M, pH 5.0), a selective, quenched fluorescence intensity was observed in the presence of Hg²⁺ amongst the sixteen cations, fourteen anions and natural organic matter humid acid analysed. The fluorescence titration data gave the analytical detection limit as 32 nM, meaning the probe could effectively monitor Hg²⁺ in the nanomolar concentration range. The probe displayed a linear response proportionally (0.1–100 μM) was reversible in its sensing mode and could track Hg²⁺ in river water samples.

Geng et al. reported a novel water-soluble polymer, Hg-62 (Fig. 54), prepared from linking a poly (vinyl alcohol) (PVA) with rhodamine B hydrazide and hexamethylenediisocyanate (Geng et al. 2014). The compound was successfully employed as both colourimetric and fluorescent probe for sensitive and selective tracking of Hg²⁺ in the mutual presence of other various alkali, alkaline earth, and transition cations, viz Co²⁺, Ag⁺, Cd²⁺, Ni²⁺, Pb²⁺, Cr³⁺, Cu²⁺, Fe³⁺, K⁺, Ba²⁺, Mg²⁺, Fe²⁺, and Zn²⁺ ions in aqueous media. The pH range 5.57–11.09 was found to be the stable pH range at which the probe works most effectually. Hg²⁺ addition to Probe Hg-62′s solution led to a notable visual colour change (from colourless to pink) and a great amplification of Probe Hg-62′s fluorescence intensity (an 11-fold enhancement) compared with the metal ions that imposed almost zero effect. The sensing of the analyte ion occurred just after a short period of about 5 min after the addition of Hg²⁺. The linear range of detection of Hg²⁺ by the synthesised compound Hg-62 was (0–8.0) × 10^–5 mol/L and the corresponding detection limit was calculated to be 5.72 × 10^–7 mol/L.

Fig. 54

Structure of Probe Hg-62 (Geng et al. 2014)

Li and the members of his team reported the sensing properties of a conjugated polymer, Probe Hg-63 (Fig. 55), which demonstrated its ability to sequester Hg²⁺ in the midst of several other cations like Li⁺, Ba²⁺, Hg²⁺, K⁺, Na⁺, Mg²⁺, Pb²⁺, Fe²⁺, Co²⁺, Ca²⁺, Mn²⁺, Fe³⁺, Cu²⁺, Cd²⁺, Cr³⁺, Ni²⁺, Al³⁺, and Zn²⁺ (Ding et al. 2017). Analysis of the fluorescence properties of Probe Hg-63 in THF showed that these cations had no influence on the fluorescence of the probe except for Hg²⁺, which showed an 11.8-fold increment. Probe Hg-63′s detection limit for Hg²⁺ tracking was calculated to be 1.0 × 10^–6 M.

Fig. 55

Structure of Probe Hg-63 (Ding et al. 2017)

 The successful report of a conjugated polymer which took advantage of aggregation-induced emission enhancement (AIEE) for the sensing of Hg²⁺ was documented by Shan and colleagues (2018). The probe’s fluorescence intensity was suppressed upon the addition of Hg²⁺ in the midst of common cations like Ag⁺, Cr³⁺, Al³⁺, Fe³⁺, Ca²⁺, Ni²⁺, Co²⁺, Pb²⁺, Cu²⁺, Zn²⁺, Mg²⁺, Fe²⁺, Mn²⁺, Cd²⁺, Ba²⁺, Li⁺, Na⁺ and K⁺, explained as due to the strong interaction of Hg²⁺ with the thioketal groups in the probe moiety. The analytical detection limit for the sensing operation was 2.3 × 10⁻⁷ mol L⁻¹.

Hg ²⁺ probes based on metal-organic frameworks (MOFs) and covalent-organic frameworks (COFs)

In 2016, Ding’s group reported a novel, superb thioether-functionalised hydrazone-linked fluorescent covalent-organic framework, COF-LZU8, with superior Hg²⁺ recognition property unequalled by many other thioether-functionalised probes (Ding et al. 2016). The report of the probe was the very first instance of the application of COF in tracking and removing cations—the sensing operation took advantage of the extended π-conjugation framework which is capable of enhancing the fluorescence signal intensity. Insight into the strong interaction between the sulphur atoms of the thioether groups in the probe moiety and Hg²⁺ was furnished by X-ray photoelectron spectroscopy and solid-state NMR. Real-time fluorescence response towards various analytical cations in acetonitrile showed that the probe effectively monitored Hg²⁺ by displaying a quenched fluorescence with a corresponding colour change.

A metal-organic framework (MOF)-based probe developed from butyne-functionalised UiO-66@Butyne (UiO-66@Butyne was synthesised by reacting terephthalic acid having two side chains to generate Butyne functionalisation, BDC@butyne, and then, MOF formation was carried out in the solvothermal method with ZrCl₄ in N,N-dimethylformamide as the solvent) was employed as a very selective and sensitive probe for tracking Hg²⁺ (Samanta et al. 2018). The probe experienced a quenched fluorescence towards Hg²⁺ in the presence of other metal ions (Ba²⁺, Cd²⁺, Co²⁺, Cu²⁺, Zn²⁺, Cr³⁺, Fe³⁺, Mg²⁺, Mn²⁺, Ni²⁺, and Sr²⁺) in an aqueous medium. None of these metal ions modulate the fluorescence intensity of the reported probe, affirming the singular response of the probe towards Hg²⁺. This sole detection of Hg²⁺ is ascribed to the oxymercuration reaction between UiO-66@Butyne and Hg²⁺ in H₂O, leading to the formation of UiO-66@OH, which displays a comparatively lesser fluorescence than the probe. The probe effectively monitored Hg²⁺ at levels as low as 10.9 nM.

A new cluster-based metal-organic framework [Hg-64, (Me₂NH₂)Cd₃(OH)(H₂O)₃(TATAB)₂](DMA)₆ (DMA = N,N-dimethylacetamide)] was synthesised for Hg²⁺ monitoring (Liu et al. 2018). As shown in Fig. 56, the probe anchors [(Me₂NH₂)Cd₃(OH)(H₂O)₃(TATAB)₂](DMA)₆ (DMA = N,N-dimethylacetamide) with triazine backbones and uses the 4,4′,4″-s-triazin-1,3,5-triyltri-p-aminobenzoic acid (H₃TATAB) as the organic spaces. The probe sensitively and selectively recognised Hg²⁺ in the presence of other cations in an aqueous medium (3 mL) via an ‘on–off’ mode and a red shift of the emission peak. Figure 57 gives the X-ray diffraction patterns, which supported the chelating interaction of the probe with Hg²⁺.

Fig. 56

Possible chelating coordination mode for the Hg²⁺ ion in Hg-64 (Liu et al. 2018)

Fig. 57

Powder X-ray diffraction (PXRD) patterns of Hg-64 (Liu et al. 2018)

Probe Hg-65 (Fig. 58) was developed from metal-organic frameworks (MOF) and was favourably engaged for the monitoring of Hg²⁺ in a sensitive, selective, and reversible manner (Wu et al. 2015). The cadmium-based probe system was the first example of a ratiometric probe constructed from MOF. In the competitive environment of other cations, only Hg²⁺ led to the quenching of the probe’s fluorescence; there existed only minimal interference in the sensing of Hg²⁺ by other cations under the same testing condition in an aqueous medium. Interesting was that the probe displayed a very minuscule detection limit of 2 nM.

Fig. 58

Structure of MOF Probe Hg-65 showing: a Cd²⁺ ions’ coordination environment in Hg-65, whose building unit is Cd-EDDA, where EDDA = ethane-1,2-diylbis(oxy)]diisophthalic acid, b the crystal packing view of Hg-60 along the ‘a’ direction, and c the channel structure of Hg-65 (Wu et al. 2015). Copyright (2015) American Chemical Society

Mercuric (II) (Hg ²⁺ ) Nanoprobes

The use of carbon dots (CDs) as ratiometric fluorescent probes was reported by Li’s group ( 2019). All detection operations were done at pH = 2.5. The ratiometric fluorescence intensity at F₄₁₄/F₅₆₅ exhibits broad linearity in the range of 30–60 μM. The detection limit of 60 nM was calculated with a rapid response time of 30 s obtained. Bano and co-workers reported carbon quantum dots as sensitive nanoprobes for Hg²⁺ monitoring via an ‘on–off’ mode (Bano et al. 2018). All detection operations were done at pH = 7 in the presence of co-existing metal ions. The detection limit of 6 nM was calculated, and the potential of the probe’s real-time monitoring of Hg²⁺ in pond water samples was determined.

The excellent affinity property of thymine with Hg²⁺ was exploited in the design of novel, water-soluble, thymine-modified graphitic carbon nitride quantum dots, T-gCNQDs, which are employed as a fluorescent nanoprobe for Hg²⁺ detection (Achadu and Revaprasadu 2018). Thymine functionalisation amplified the fluorescence of the QDs, and the interaction of thymine with Hg²⁺ through the formation of a T-Hg²⁺-T complex gives the basis for Hg²⁺ tracking. Optical detection in phosphate buffer (10 mM, pH 7.2) resulted in the dampening of the fluorescence intensity of the probe by Hg²⁺ in the joint existence of other cations (Ca²⁺, Ni²⁺, Cu²⁺, Fe²⁺, Co²⁺, Mn²⁺, Pb²⁺, Al³⁺, Na⁺, K⁺, Cd²⁺, Cr³⁺, Mg²⁺, and Ag⁺). Broad linearity of detection (1.0–500 nM) was observed with a detection limit of 0.15 nM calculated. The probe is applicable for Hg²⁺ monitoring in tap and pond water samples.

A colourimetric probe designed based on the Hg²⁺-induced deprotection and morphology transition of 1-dodecanethiol (C₁₂H₂₅SH)-capped silver nanoprisms (Ag NPRs) in the presence of I⁻ at ambient temperature was reported by Chen et al. (2013a). The reported compound was unresponsive to pH changes in the range of 3.6–5.9. In HAc/NaAc buffer solutions (10 mM, pH 5.0), the probe detected Hg²⁺ sensitively and selectively with a magnificent 100-time fluorescence intensity increment (‘turn on’) noticed upon the addition of Hg²⁺, whereas the addition of the other cations like Fe²⁺, Cu²⁺, Zn²⁺, Mn²⁺, Cd²⁺, Pb²⁺, Mg²⁺, Ba²⁺, Co²⁺, and Ag⁺ induced a rather benign influence on the probe’s fluorescence intensity under the same optimised testing conditions. The upward shift of the fluorescence intensity was explained as due to the presence of a strong affinity between the thiol groups in the probe molecule and Hg²⁺. A rapid response time of 15 min (which showed stability even up to 30 min) was calculated for the detection of Hg²⁺ by the designated probe. A linear relationship between the increasing additions of Hg²⁺ to the probe’s solution was proposed, and the calculated detection limit was 3.3 nM. Interestingly, the probe was employed for Hg²⁺ tracking in drinking water, tap water, and pond water samples with excellent recoveries achieved.

Huang et al. designed and synthesised a water-soluble fluorescent probe, based on fluorescence quantum dot (Mn: CdS/ZnS) functionalised 33-mer thymine-rich single-stranded DNA (strand A) and gold nanoparticles (GNPs) functionalised 10-mer single-stranded DNA (strand B), for Hg²⁺ sensing (Huang et al. 2013b). In 0.01 M PBS buffer, the initially weak fluorescence intensities of strands A and B experienced dramatic upward catapults (‘turn on’) upon Hg²⁺ addition (1.0 μM) in the sample solution, whilst the other cations like Mn²⁺, Ba²⁺, Ni²⁺, Cu²⁺, Ca²⁺, Cr²⁺, Co²⁺, Cd²⁺, Mg²⁺, Zn²⁺, Al³⁺, Fe³⁺, Pb²⁺, Ag⁺, and Au³⁺ (10 μM) left no influence on the probe’s fluorescence intensity. The authors showed that the designated probe responded to Hg²⁺ detection within two linear ranges, i.e. (0–1) × 10^–9 M and 1 × 10^–9 – 1 × 10^–8 M. The calculated detection limit was obtained down to 0.18 nM. The probe demonstrated reversibility in its detection property, and ultimately, it was successfully applied for the tracking of Hg²⁺ both in tap and lake water samples.

Huang and co-workers constructed a simple, green fluorescent nitrogen-doped carbon nanoparticles (FNCPs)-based probe, using strawberry juice as the source of carbon (Huang et al. 2013c). The reported probe that bears both carboxylic and amino groups showed ultra-high sensitivity and selectivity for Hg²⁺ via fluorescence quenching. The linear ranges of Hg²⁺ detection fell within two broad ranges: 10–100 nM and 1–50 µM. It was shown that the monitoring of Hg²⁺ by the designed compound was complete at about 10 min. In 1 mL of phosphate solution (25 mM, pH 7.4), the addition of Hg²⁺ to the probe’s solution engendered a massive quenching of the fluorescence intensity (‘turn off’) in the midst of 15 cations such as Ca²⁺, Ag⁺, Ni²⁺, Cr³⁺, Al³⁺, Cu²⁺, Ba²⁺, Fe³⁺, Pb²⁺, Zn²⁺, Fe²⁺, Co²⁺, Mg²⁺, Mn²⁺, or Cd²⁺. Meanwhile, the other various tested cations left the fluorescence intensity unchanged. The calculated limit of detection (LOD) was 3 nM. The practical usage of the probe was demonstrated successfully as given by its efficacy to measure Hg²⁺ concentration in lake water samples.

The preparation of the water-soluble, ‘green’ fluorescent carbon nanoparticles (CPs)-based probe that uses sweet potatoes as the carbon source was reported by Lu et al. (2012). The probe was obtained via the hydrothermal technique and houses both hydroxyl and carboxylic or carbonyl moieties in its architectural skeleton. The employment of the probe for the sensitive and selective monitoring of Hg²⁺ via a fluorescence dampening mode was envisaged. The best working pH range of the described probe was 1.0–7.0. The detection of Hg²⁺ traversed over two linear concentration ranges of 0.5–10 and 500–4 × 10⁴ nM, and the calculated detection limit was 0.23 nM. It was proposed that electron or energy transfer was responsible for the quenching phenomenon of the probe’s fluorescence intensity (‘turn off’) observed by Hg²⁺ addition in PBS (pH 7.0; λ_ex = 360 nm) when jointly existent with other cations such as Ag⁺, Ca²⁺, Fe³⁺, Co²⁺, Cd²⁺, Cu²⁺, Mg²⁺, Mn²⁺, Pb²⁺, Ni²⁺, and Zn²⁺, which impinged no monumental influence on the probe’s fluorescence intensity. The sensing of Hg²⁺ by the CPs probe was successfully recovered by the addition of Cys. As a proof of concept, the efficaciousness of the CPs probe for Hg²⁺ tracking in the real environmental system was successfully demonstrated in lake water samples.

Lu’s group documented bovine serum albumin (BSA), assembled in a silver nanocarbon (BSA–Ag NCs), which functions as a novel ratiometric fluorescence probe for the monitoring of Hg²⁺. Experimental results furnished excellent linearity of the fluorescence response for the tracking of Hg²⁺ within the concentration range of 0–2.5 mg mL⁻¹ (Lu et al. 2013). Meanwhile, the detection limit of 4.87 × 10⁸ M was obtained from the Benesi–Hildebrand plot analysis, a much lower value than those of ratiometric fluorescent probes of organic origins. Upon the addition of Hg²⁺, the fluorescence intensity of the reported compound at 634 nm was reduced (‘turn off’); an elevation at 480 nm with a concomitant colour variation from light yellow to colourless and a sharp red to blue fluorescence was observed. The anti-interference property of the designed Ag NC probe compound was good, implying that the monitoring of Hg²⁺ by the probe was not significantly interfered by several other cations such as Na⁺, Ag⁺, Ca²⁺, Co²⁺, Cd²⁺, Ni²⁺, Mg²⁺, Pb²⁺, Cu²⁺, Zn²⁺, Al³⁺, and Fe³⁺ investigated, with the exception of Ag⁺ that induced a minor disturbance.

Paramanik et al. designed an Au nanocluster–CdTe quantum dots nanocomposite probe, utilised for the recognition of Hg²⁺ (Paramanik et al. 2013). Desirably, the addition of cations like Co²⁺, Fe³⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pb²⁺, Sr²⁺, Zn²⁺, Cd²⁺, Ca²⁺, and Cu²⁺ did not leave any meaningful impact on the fluorescence intensity of the reported probe, except for Hg²⁺ that quenched the fluorescence dramatically (‘turn off’) (about 74%-fold) at 553 nm. Although Cu²⁺ impinged a 26% quenching of the probe’s fluorescence, the effect was rather insignificant compared to that posed by Hg²⁺. The calculated detection limit of Hg²⁺ recognition by the probe is 9 nM.

Singh et al. constructed a new Biginelli-based organic nanoparticle-based compound (Singh et al. 2013). The designated molecule, Probe Hg-66 (Fig. 59), which employs both a conformational change and an intramolecular charge-transfer process, was used as a fluorescent probe for the sensitive and selective monitoring of Hg²⁺ in an aqueous medium. In Tris buffer (pH = 7.4, 1 mM) solution, the sequential addition of Hg²⁺ to the solution of the probe suppressed its fluorescence intensity massively (‘turn off’) in the co-presence of other rival cations investigated. The limit of detection was obtained down to 1 nM. The response time for Hg²⁺ detection by the probe was 3 min.

Fig. 59

Structure of Biginelli-based organic nanoparticle-based Probe Hg-66 (Singh et al. 2013)

The usage of mercury-specific DNA (MSD-DNA)-functionalised gold nanoparticles (AuNPs), as probe for Hg²⁺ tracking was described by Tan’s group (Tan et al. 2013). The concentration range of Hg²⁺ detection by these probe molecules was determined to be 0.02–1.0 μM. Experimental results revealed a lowering of the fluorescence intensity of the probe (‘turn off’) upon the incremental addition of Hg²⁺ in the presence of other cations (including Co²⁺, Fe³⁺, Mn²⁺, Ca²⁺, Mg²⁺, Cr³⁺, Cu²⁺, Ni²⁺, and Pb²⁺) assessed. The sensing experiments furnished the limit of detection of as 16 nM. Noteworthy is that the monitoring of Hg²⁺ in actual river water samples was realised by the synthesised AuNPs–MSD-based probe.

Inspired by a time-gated fluorescence resonance energy transfer (TGFRET) detection technique, Huang and his team members again attained the fluorescence detection of Hg²⁺ by employing water-soluble ssDNA functionalised quantum dots (which constituted the energy-transfer donor unit) and gold nanoparticles (which served as the energy-transfer acceptor moiety) (Huang et al. 2013a). The analysis of the experimental results showed that there was a significant quenching of the reported compound’s fluorescence intensity (‘turn off’) in a linear fashion (i.e. 1 × 10^–9–1 × 10^–8 M) upon the addition of varying Hg²⁺ concentrations. By contrast, the addition of various investigated cations left an insignificant effect on the fluorescence intensity of the compound. Moreover, a suitably low detection limit of 0.49 nM or 0.1 ppb was obtained. Desirably, the compound was shown to successfully track Hg²⁺ in tap water, river, and lake water samples with excellent results achieved.

Via a hydrothermal treatment, Li et al. developed water-soluble nitrogen-doped carbon quantum dots (N-CQDs) for the detection of Hg²⁺ through selective and sensitive fluorescence dampening approach (Li et al. 2015). The fluorescence intensities of the developed N-CQDs experienced stability in the broad pH range of 4.0–10.0. N-CQDs’ fluorescence was invariably unperturbed upon the addition of other cations of seventeen different kinds, except for a large fluorescence suppression caused by Hg²⁺ (‘turn off’). The linear concentration range of Hg²⁺ detection was from 10 nM–20 mM, with a detection limit of 8.60 nM or 1.72 ppb estimated. The probe’s lost fluorescence intensity was regained (about 79-fold regeneration) after I⁻ was added in the presence of ten other different anions. This result demonstrated the ability of the probes to also monitor I⁻ within a linear detection range of 0.50 mM– 40 mM or 63.50–5080 mg L⁻¹ observed with a detection limit of 0.354 mM or 45.0 mg L⁻¹ calculated.

Li and his research group gave an account of a water-soluble graphene oxide (GO) probe, functionalised with single-stranded DNA aptamer, and employed for a label-free Hg²⁺ tracking via a fluorescence quenching phenomenon (Li et al. 2013). In PBS solution (10 mM Na₂HPO₄/NaH₂PO₄, 0.3 M NaCl, 0.1 mM ethylenediamine, pH = 7.0), the probe’s initially strong fluorescence intensity at 488 nm excitation wavelength was largely and concomitantly suppressed (‘turn off’) upon the addition of Hg²⁺ when several cations like Ag⁺, K⁺, Cd²⁺, Ca²⁺, Cu²⁺, Ni²⁺, Pb²⁺, Co²⁺, and Fe³⁺ were present. The linear tracking range of the probe was noted to be from 1 nM–50 nM, and the quenching phenomenon was observed to lapse only for ca. 5 min. The estimated detection limit was sufficiently low down to 0.92 nM or 0.18 ppb.

The works of Zhang et al. (2015b) described a sensitive, fluorometric labelled ss-DNA for tracking Hg²⁺. The fluorescence increment in the developed bioassay was induced by graphene oxide (GO) and exonuclease (exo) III. The detection limit and response time of the assay were calculated to be 0.1 nM and 8 min, respectively. Mediated by Hg²⁺, there was a formation of a well-folded dsDNA by the FAM-labelled ssDNA which became digested by Exo III, leading to the release of FAM-labelled mononucleotide and Hg²⁺. The FAM-labelled mononucleotide was not accumulated onto the GO surface, resulting in increased fluorescence intensity. The released Hg²⁺ then induced a new cycle of this whole process. In this way, a minuscule quantity of Hg²⁺ can engender a plethora of enzyme digestion reactions and form a massively amplified fluorescence signal. The assay could excellently monitor Hg²⁺ in lake water samples. 

For a collection of other nanomaterial-based probes developed for Hg²⁺ monitoring, readers are referred to the brilliant review article by Duan and Zhan (2015).

Other sterling instances of the report of fluorescent, colourimetric, and/or ratiometric probes for Hg²⁺ monitoring exist in the literature, but owing to space limitation, these are reserved for the author’s future review assignments.

Important generalisations

From the foregoing careful discussion on various kinds of fluorescent, colourimetric, and/or ratiometric Hg²⁺-selective probes developed from diverse fluorophore motifs, these important generalisations are made:

• Probes such as Probes Hg-1, Hg-7, Hg-8, and Hg-13 appended with solubilising groups like the amino group, –NH₂, tend to be more soluble in water than those without this functionality. Moreover, amino group-appended probes possess enhanced quantum yield than their counterparts without this functional group.

• For ages, there has been a heavy reliance on the use of nitrogen-centric motifs for the construction of Hg²⁺ probes, but the need for nitrogen coordination for signalling processes usually encounters some inadequacies. Besides an exclusive reliance on nitrogen coordination chemistry, there is always a possibility of false-positive signalling owing to protonation or pH sensitivity. This limitation has been largely tackled by a research focus shifting towards the employment of sulphur-incorporating groups, although this also is not without its restraints.

• Hg²⁺ being a soft Lewis acid and equally possessing a strong thiophilic affinity has prompted many reported Hg²⁺-selective probes typically to employ sulphur as a soft donor atom to facilitate a tight binding to the metal as part of the fluorescence probe architectures. Reported probes that employ this strategy include Probes Hg-4, Hg-6, Hg-7, to mention a few. Only a few probes for Hg²⁺ based on alkynes have been developed according to the π electrophilicity of Hg²⁺. Furthermore, their signalling modes are due to the irreversible chemical reactions of alkynes as opposed to reversible interactions. This impedes their practical applications. As a basic requirement, ideal probes should possess a reversibility property (Dong et al. 2017).

• Most longstanding strategies employed for the design of Hg²⁺-selective probes are based on the complexation or chemical reaction between the probes and Hg²⁺. The reaction-based design includes Hg²⁺-induced desulphurisation and desulphurisation which ensues in cyclisation to the heterocycle, mercuration, and Hg²⁺-promoted hydrolysis reactions. Comprised in the latter category is a typical reaction-based strategy tagged ‘Hg²⁺-promoted deprotection of thioacetals’ which has been taken advantage of to generate a host of probes. Reported probes which makes use of this design strategy include Probes Hg-14, Hg-15, Hg-21–Hg-25, Hg-53, Hg-57, Hg-58, etc.

• There is increasing gravitation towards the use of red to near-infrared (NIR) probes (which possess > 600 nm) owing to their merits of profound tissue penetration, reduced photodamage to biological models and lowered interference ensuing from background autofluorescence of biomolecules in living systems. This is particularly relevant to the construction of rhodamine probes since the absorption and emission wavelengths of most rhodamine derivatives are < 600 nm, placing a restraint on their biological applications. Instances of NIR probes reported in this review include the case of Probe Hg-32.

• Probes, such as Probes Hg-9, Hg-32, Hg-48, Hg-56, Hg-65, and others that make use of ratiometric fluorescence signalling mode are more desirable and gaining increasing research attention than those with a single fluorescence response wavelength. Basically, ratiometric probes operate by making use of the ratio of the emission intensity at two different wavelengths. Ratiometric detection strategy eradicates uncertainties mostly due to environmental conditions like polarity, pH, temperature, and non-homogeneity of distribution of fluorescent indicators in cells. Sterling instances of such probes include the squaraines-based probes which possess long excitation and emission wavelengths (have sharp absorption around ca. 640 nm).

• Inorganic quantum dots are gaining grounds as alternative fluorophores due to their good photobleaching anti-resistance and possession of high quantum yields and wide light-absorption band. Unfortunately, inorganic quantum dots typically contain Se, Cd, Pb, or other toxic elements (Das and Tan 2008). Carbon QDs have replaced inorganic QDs as the former possess greater photostability and reduced toxicity. Recently, graphene oxide (GO), which contains carbon (being environmental-friendly, earth-abundant, and inexpensive), has emerged as a new fluorophore because of its good water solubility, strong photoluminescence, great mechanical strength, facile surface alteration, and the ability for bio-conjugation.

• A rapid response time is highly essential for either an on-site or online field monitoring. Good response times have been achieved by many of the reported probes, including Probes Hg-10, Hg-13, Hg-14, Hg-15, Hg-16, Hg-32, Hg-41, Hg-61, etc.

• In all analytical detection sense, fluorescence ‘turn on’ probes are more sought after than their fluorescence ‘turn off’ counterparts since the former usually display lower background and higher sensitivity than the latter (Wang et al. 2013).

Conclusions

In this review, fluorescent, colourimetric, and/or ratiometric probes based on the molecular motifs of synthetic small organic molecules, supramolecules, polymers, metal-organic frameworks, covalent-organic frameworks, and nanoparticles have been discussed in the context of the detection of mercuric (II) (Hg²⁺), giving recent updates within the timeframe of 2011–2019. Probes that exhibit fluorescence or colourimetric variation in response to various analytes are preferable to other types of probes, as they emit easily recognisable optical response by fluorimeters or simply by naked eyes. The main features highlighted in the reported probes in this review were the parameters of the most suitable solvent system, optimum pH value/range, interference property, detection limit, and association constant. The environmental and/or biological utilities of these probes are also discussed wherever possible. Even though research works on the detection of Hg²⁺ have been very successful thus far, there still exist a couple of lingering obstacles which need tackling for the achievement of further advancements in the science of Hg²⁺ detection.

Presently, specific instances of Hg²⁺-selective probes that possess the simultaneous qualities of supersensitivity and selectivity, rapid response time, convenience of preparation, low toxicity, and above all, water solubility are rare. Meanwhile, many organic probes require laborious synthetic methods, whilst probes based on quantum dots (QDs) exhibit toxicity. Besides, a large proportion of these probes possess very short fluorescence lifetime and utilise visible light emission which cannot be used in in vivo sensing. Other challenges are irreversible Hg²⁺ complexation, poor selectivity, and long response time. Perhaps the greater challenge, now, is the difficulty in designing and synthesising probes that could overcome all these challenges.

The spin–orbit coupling which induces a ‘heavy atom’ effect makes Hg²⁺ intrinsically a quencher of fluorescence. This phenomenon, within the convention of sole diagnostic monitoring of Hg²⁺, is undesirable owing to the reason that some other heavy cations like Cu²⁺, Pb²⁺, Cd²⁺, and Ag⁺ can also induce similar effect, interfering with the detection of Hg²⁺ and thereby leading to confusing observation and interpretation of experimental phenomena and results. As such, more ‘turn on’, interference-free Hg²⁺-selective probes should be developed (as opposed to the diverse ‘turn off’ ones that exist in several bodies of literature) by researchers in this field.

If only these considerations would be favourably addressed, more and better Hg²⁺-selective probes would be designed, developed, and reported from various labs from across the world, with the possibility that the future of Hg²⁺ detection would brighter than it is now.