Design strategies and progress on xanthene-based fluorescent probe for metal ions

Probes for Cu²⁺

Most of the xanthene hydrazone probes could be applied in the identification of Cu²⁺ because of the good affinity of nitrogenous and oxygenous recognition moiety to Cu²⁺ according to the soft-hard acid-base theory. Copper ions, a kind of transition metal ions, play an essential role in a variety of fundamental physiological processes in organisms ranging from bacteria to mammals, and its distribution is strictly controlled in vivo. On the one hand, excess copper can cause a highly toxic state and lead to serious infant liver damage, such as Wilson’s disease. On the other hand, the loss of copper homeostasis can result in Alzheimer’s disease and Menkes disease. A comprehension of the physiological and pathological functions of Cu²⁺ in living cells is very significant (Liu et al. 2012b, Ge et al. 2013b, Goswami et al. 2014). Therefore, fluorescent probes for the detection of copper ion have been widely explored. Among them, the most interesting, selective, and sensitive probes are those that are based on xanthene hydrazone (Figure 3).

Figure 3:

Probes 1–20 based on xanthene hydrazone for detecting Cu²⁺.

In 2011, Zhao et al. described the development of a rhodamine chromene-based fluorescence probe 1 to monitor the intracellular Cu²⁺ level in living HeLa cells, which exhibited a fluorescence response toward Cu²⁺ under physiological conditions with high sensitivity and selectivity (Liu et al. 2011). The fluorescence intensity was significantly increased by about 40-fold with 10 equivalents of added Cu²⁺. The data from Job’s method exhibited a maximum absorbance when the molecular fraction of 1 was close to 50%, which also suggests a 1:1 stoichiometry for the 1-Cu²⁺ complex (Figure 4).

Figure 4:

Recognition mechanism of probe 1 with Cu²⁺.

In the same year, a new probe 2 based on the spirolactam form of rhodamine 101 hydrazone was reported by Xie’s group, which exhibited a highly selective and sensitive response toward Cu²⁺ in aqueous solutions (Xie et al. 2011). The structural rigidity introduced by multiple n-propylene bridges of rhodamine 101 moieties can shift the absorption and emission maxima to longer wavelengths, with higher molar extinction coefficients and higher quantum yields compared to rhodamine B. The fluorescence of probe 2 can be detected in a low concentration (2×10⁻⁶ mol/l) of Cu²⁺. The results revealed that probe 2 is a good colorimetric probe to Cu²⁺ in the red region (Figure 5).

Figure 5:

Recognition mechanism of probe 2 with Cu²⁺.

8-Hydroxyquinoline-based ligands with extended conjugated fluorophores can form luminescent chelates with a number of metal ions. Therefore, a novel rhodamine-quinoline derivative-based probe 3 was designed and synthesized by Feng et al. which exhibited highly selective and sensitive colorimetric and “turn-on” fluorescent responses toward Cu²⁺ ions in aqueous solution (Feng et al. 2012). The good linear relationship was easily obtained from the fluorescence changes and the concentrations in the range of 20–120 mm. The fluorescence change was remarkable for the Cu²⁺ ion detection even in the presence of other metal ions, such as Ca²⁺, Cd²⁺, Co²⁺, Hg²⁺, Ni²⁺, Zn²⁺, Ba²⁺, Mg²⁺, and Pb²⁺.

In 2012, a new probe 4 based on rhodamine B with 1,2,4-triazole as subunit was synthesized and characterized by Zhang (Zhang et al. 2012). Because of the 1,2,4-triazole subunit containing lone electron pairs on N, the semirigid ligand could effectively chelate Cu²⁺ according to the ionic radius and limit the geometric structure of the complex. Probe 4 exhibited high selectivity and sensitivity for Cu²⁺ in ethanol/water (6:4, v/v) of pH 7.0 HEPES buffer solution and underwent ring opening mechanism, and a 2:1 metal-ligand complex was formed. Probe 4 displayed a linear response to Cu²⁺ in the range between 1.0×10⁻⁷ and 1.0×10⁻⁶m with a detection limit of 4.5×10⁻⁸m. Its capability of biological application was also evaluated and the results showed that probe 4 could be successfully employed as a Cu²⁺-selective probe in the fluorescence imaging of living MCF-7 cells (Figure 6).

Figure 6:

(A) Recognition mechanism of probe 4 with Cu²⁺ and (B) confocal fluorescence images in MCF-7 cells. Reprinted from Zhang et al. (2012).

Zhao et al. described the development of a rhodamine chromene-based “turn-on” fluorescence probe 5 to monitor the intracellular Cu²⁺ level in living cells (Liu et al. 2012a). The new fluorescent probe with a chlorine group in chromene moiety exhibited good membrane-permeable property because the predicted lipophilicity of the present probe is strong, and a fluorescence response toward Cu²⁺ under physiological conditions with high sensitivity and selectivity facilitates the naked-eye detection of Cu²⁺ (Figure 7). The fluorescence quantum yield of 5 with 15 equivalents of Cu²⁺ was 0.26 at an excitation wavelength of 530 nm, which was higher than what was previously reported. The results showed a linear response range covering a concentration range of Cu²⁺ from 0.1×10⁻⁵ to 7.0×10⁻⁵m and the correlation coefficient R is 0.9896. The detection limit was 2.3×10⁻⁷m. The fluorescence intensity was remarkably increased upon the addition of Cu²⁺ within 1 or 2 min, whereas the other 16 metal ions (Na⁺, Mg²⁺, Al³⁺, K⁺, Ca²⁺, Cr³⁺, Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Hg²⁺, Zn²⁺, Ba²⁺, Pb²⁺, Ag⁺, and Cd²⁺) caused no significant effect.

Figure 7:

Recognition mechanism of probe 5 with Cu²⁺.

In 2013, Thennarasu et al. reported two new rhodamine-indole conjugates 6 and 7 (Chereddy et al. 2013b). They have provided evidence to show that both the sensitivity and the selectivity of probes 6 and 7 were tunable by choosing appropriate solvent systems. Compared to probe 6, probe 7 had higher selectivity and sensitivity for Cu²⁺ and had excellent stability in physiological pH conditions. The limit of detection of Cu²⁺ (3×10⁻⁸m) was lower than the values reported for several other rhodamine-based Cu²⁺ probes. Moreover, probe 7 is not toxic up to ~10 mm and can be used for the detection of Cu²⁺ in live fibroblast cells. To explore the importance of the chelating ring size, nature, and orientation of coordinating atoms in the metal ion selective complex formation, they further synthesized three fluorescent probes 8–10 using a similar strategy (Chereddy et al. 2013a). The electrospray ionization-mass spectrometry (ESI-MS) data of the Cu²⁺ complexes of probes 8 and 9 suggested a 1:2 stoichiometry for both complexes. However, as the triazole (pK_a=0.3) is slightly more basic than pyridine (pK_a=5.2), probe 10 containing a triazole group instead of the pyridine group should favor the formation of the 10-Cu²⁺ complex in a 1:1 stoichiometry. Meanwhile, the interference from Fe³⁺ ions observed in the cases of probes 8 and 9 was not observed in the case of probe 10. Therefore, probe 10 is highly selective toward Cu²⁺, forms a 1:1 Cu²⁺-complex, and allows the spectrometric as well as naked-eye detection of Cu²⁺ in aqueous and biological samples. It is useful for the detection of intracellular Cu²⁺ levels in live keratinocyte cells because probe 10 is stable at physiological pH and is nontoxic to NIH 3T3 cells at low concentrations (Figure 8).

Figure 8:

Fluorescent images of live keratinocyte cells incubated with probes 7 and 10. Reprinted from Chereddy et al. (2013a,b).

Based on the FRET mechanism, Das et al. have synthesized a new indole functionalized rhodamine probe 11, which binds to Cu²⁺ with visually observable changes in their electronic and fluorescence spectral behavior (Kar et al. 2013). Probe 11 emitted only weak fluorescence at 490 nm when excited at 340 nm, whereas it resulted in a decrease in the fluorescence intensity at 490 nm and an increase in the fluorescence intensity at 587 nm upon the addition of Cu²⁺ to a solution containing 11 (Figure 9). These spectral changes are significant enough in the near-infrared (NIR) and visible regions of the spectrum and thus enable naked-eye detection. The spectral change is due to the formation of the 11-Cu complex and the deprotonation of the indole unit occurs simultaneously with the addition of Cu²⁺. The apparent binding constant for the formation of 11-Cu complex was calculated by considering a 1:1 binding stoichiometry. Studies manifested that 11-Cu complex is selective and fully reversible in the presence of sulfide anions. As determined by confocal fluorescence microscopy, probe 11 could also be used as an imaging probe for the detection of Cu²⁺ in HeLa cells.

Figure 9:

Cu²⁺-induced FRET OFF→ON of probe 11.

In 2013, Long et al. developed a rhodamine-based fluorescent probe 12 for Cu²⁺ bearing the 8-hydroxyquinoline unit (Wang et al. 2013). Probe 12 formed a 1:1 complex with Cu²⁺ by cooperating the O and N of the 8-hydroxyquinoline unit and carbonyl O and N of rhodamine hydrazide unit. Therefore, it exhibited favorable features, including reversibility, high sensitivity with a large fluorescence enhancement (80-fold) and a low detection limit of 0.19 μm, high selectivity for Cu²⁺ over other heavy and transition metal (HTM) ions in Tris-HCl/ethanol (7:3, v/v, pH 7.4), working well at physiological pH, and good cell membrane permeability. Finally, probe 12 can be applied in the imaging of Cu²⁺ in living cells (Figure 10).

Figure 10:

Fluorescence images of MG-63 cells. Reprinted from Wang et al. (2013).

Tang et al. (2013) reported the synthesis and photophysical properties of a new rhodamine B-based probe 13, which exhibited high selectivity, sensitivity, and rapid recognition behavior toward Cu²⁺ via colorimetric and fluorescent detection mode. The detection limit of probe 13 was evaluated to be 7.96×10⁻⁸m. The Cu²⁺ recognition process is reversible and barely interfered by other coexisting metal ions, including Hg²⁺, Ag⁺, Pb²⁺, Sr²⁺, Ba²⁺, Cd²⁺, Ni²⁺, Co²⁺, Fe²⁺, Fe³⁺, Mn²⁺, Cu²⁺, Zn²⁺, Al³⁺, Cr³⁺, Mg²⁺, K⁺, and Na⁺.

In 2014, Dong et al. synthesized a novel rhodamine fluorescent probe 14 by reacting rhodamine B hydrazide with 3-bromo-5-methylsalicylaldehyde, which has been developed as a new colorimetric probe for the selective and sensitive detection of Cu²⁺ (Zhang et al. 2014). Experimental results indicated that probe 14 can provide a rapid, selective, and sensitive response to Cu²⁺ with a linear dynamic range from 0.667 to 240 μmol/l. Common interferential ions did not show any interference on Cu²⁺ determination. It was anticipated that probe 14 can be a good candidate probe and has potential applications for Cu²⁺ determination. Besides, the proposed probe 14 exhibited the following advantages: a quick, simple, and facile synthesis (Figure 11).

Figure 11:

Recognition mechanism of probe 14 with Cu²⁺.

In 2015, an “off-on” rhodamine-based fluorescent probe 15 for the selective detection of Cu²⁺ has been designed and synthesized by Mao et al. which showed a highly Cu²⁺-selective fluorescence enhancement response in an aqueous pH 7 (Mao et al. 2015). Under optimum conditions, the increase of fluorescence intensity was linearly proportional to the concentration of Cu²⁺ over a wide range, and the limit of detection was 29 nm. The study indicated that a 1:1 stoichiometry complex was obtained by the copper ion chelated with vanillin-O, carbonyl-O, and rhodamine 6G hydrazide-N (Figure 12). Therefore, the present method could be applied for the analysis of Cu²⁺ in an aqueous system.

Figure 12:

Recognition mechanism of probe 15 with Cu²⁺.

Yang et al. reported a damantane-modified salicylrhodamine B and β-cyclodextrin-modified Fe₃O₄@SiO₂ assembled by host-guest interactions, which induced novel inclusion complex magnetic nanoparticles (SFIC MNPs) colorimetric sensitive for Cu²⁺ (Zhang et al. 2015b). Probe 16 exhibited a clear color change from colorless to pink selectively and sensitively with the addition of Cu²⁺ in the experiments of UV-visible spectra, and the detection limit was measured up to 5.99×10⁻⁶m in solutions of CH₃CN/H₂O (1:10). The SFIC MNPs were superparamagnetic according to magnetic measurements and could be separated and collected easily with a commercial magnet in 9 s. In addition, microspheres have also shown a good ability of separating for other ions from aqueous solutions due to a large number of hydroxyl groups on the surface (Figure 13).

Figure 13:

Chemical and schematic illustration of the preparation of SFIC MNPs probe 16 for Cu²⁺.

Levels of lysosomal copper are closely related to the human body. However, there is one major limitation of detection methods for monitoring dynamic changes in copper pools that are unable to diagnostically assess the influence of copper accumulation on health status. Fortunately, Yang et al. reported a rhodamine fluorescent probe 17 activated by the presence of lysosomal Cu²⁺ in 2015 (Li et al. 2015b). Upon activation by lysosomal acidic pH, probe 17 bound with Cu²⁺ by spiropyran-based proton activation, promoting rhodamine ring-opening, which showed a “switched on” fluorescence signal (Figure 14). This strategy resolved some common challenges of chemical probes in biosensing, such as spatial resolution in cell imaging, solubility and stability in biological system, and interference from intracellular species. The new design, allowing one to track on a location-specific basis and visualizing lysosomal Cu²⁺, could offer a potentially rich opportunity to examine copper physiology in both healthy and diseased states.

Figure 14:

Recognition mechanism of probe 17 with Cu²⁺.

In 2016, a new rhodamine probe 18 with a high selectivity for Cu²⁺ was synthesized by Wang et al. (Zhang et al. 2016a). The analytical results obtained by UV-vis and fluorescence spectrophotometry show that the linear range of probe 18 for Cu²⁺ is 0.50–20.00 and the limit of detection is 0.11 μm. The 1:1 stoichiometric structure between probe 18 and Cu²⁺ could be formed because the copper ions are cooperated with the O atom of introduced moiety, O atom of carbonyl, and N atom of rhodamine B hydrazide. Meanwhile, it can be easily seen by the naked eye that the pink color of a 18-Cu²⁺ solution immediately disappeared after the addition of excess sodium EDTA, with the fluorescence intensity returning to the original state. Probe 18 can be employed as a reversible fluorescent probe for Cu²⁺ in drinking water and living HeLa cells.

In 2013, our group reported three new rhodamine Schiff base probes 19–21 (Yang et al. 2013b). The probes displayed remarkably Cu²⁺-selective orange fluorescence and pink color switch over a wide range of tested metal ions in ethanol-PBS (5/5, v/v, pH 7.4) solution. Job’s method was applied to study the binding stoichiometry of 19 and Cu²⁺. The 2:1 stoichiometry is the binding mode of the probe and Cu²⁺ because the introduced cinnamyl aldehyde has no heteroatom. The coordination was also investigated to be a reversible process because the fluorescence disappeared when excess ethylenediamine was added to the colored solution of complex. Confocal laser scanning microscopy experiments revealed that the probes demonstrated the value for sensitive and selective detection of intracellular Cu²⁺ in living HeLa cells (Figure 15).

Figure 15:

Proposed mechanism for the fluorescent changes upon the addition of Cu²⁺ and fluorescent images of HeLa cells incubated with probes. Reprinted from Yang et al. (2013b).

The above-mentioned strategy was subsequently extended that we have conducted comprehensive studies to explore the structure-property relationships of these probes 19–27, which contain differently substituted cinnamyl aldehyde with a C=N Schiff base structural motif (She et al. 2016). Extensive work has been focused on the structure-property relationships of this probe model and has investigated the change of optical properties caused by different electronic effects and steric effect of the recognition group. More importantly, density functional theory (DFT) calculation simulates the ring-closed and ring-open forms of these modular Schiff base Cu²⁺ fluorescent probes. In addition, confocal laser scanning microscopy experiments suggested that all the probes would be a powerful tool for the sensitive and selective detection and mapping of Cu²⁺ adsorbed in environmental microbial systems. This approach provides a significant strategy for studying structure-property relationships and guiding the synthesis of probes with various optical properties (Figure 16).

Figure 16:

Schematic of the strategy for studying the structure-property relationships of nine modular Cu²⁺ fluorescent probes and single-cell scale maps showing the sorption of Cu²⁺ to cell-EPS-mineral aggregates. Reprinted from She et al. (2016).

Probes for detecting Hg²⁺

To date, probes containing Schiff base structural motif have been developed successfully to detect Cu²⁺. Therefore, there is a series of xanthene thiohydrazone probes that would be applied in detecting Hg²⁺ when the carbonyl oxygen atom is replaced by sulfur atom. Mercury ion is one of the most toxic metal ions. Mercury overload can cause serious damage to the brain, nervous system, endocrine system, and even the kidneys because of its toxic effects. Based on the soft-hard acid-base theory, Hg²⁺ is a representative example of soft acid and sulfur is a soft base. Therefore, a sulfur-based functional group must be a good candidate as the S is strong binding site for Hg²⁺. Based on the above reasons, xanthene thiohydrazone probes, where the carbonyl oxygen atom is replaced by sulfur atom, were designed and synthesized (Figure 17).

Figure 17:

Probes based on rhodamine thiohydrazone for detecting Hg²⁺.

In 2011, a novel fluorescent probe 28 has been reported by Jiang to detect Hg²⁺ in aqueous buffer solution, which demonstrated high selectivity for sensing Hg²⁺ with about 383-fold enhancement in fluorescence emission intensity and micromolar sensitivity (K_d=7.5×10⁻⁶ mol/l; Wang et al. 2011a). In addition, in the presence of other metal ions, such as alkali and alkaline earth metal ions (K⁺, Na⁺, Mg²⁺, and Ca²⁺) and other transition metal ions (Mn²⁺, Ni²⁺, Co²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Ag⁺, Pb²⁺, Cr³⁺, and Fe³⁺), there was no evident fluorescence intensity enhancement because Hg²⁺ could be distinguished from other metal ions. Probe 28 is cell permeable and can visualize the changes of intracellular mercury ions in living cells using fluorescence microscopy (Figure 18).

Figure 18:

Recognition mechanism of probe 28 with Hg²⁺.

In the same year, Yang et al. reported a novel rhodamine-based highly sensitive and selective colorimetric “off-on” fluorescent probe 29 for Hg²⁺ ions designed and prepared using the well-known thiospirolactam rhodamine chromophore and furfural hydrazone as signal-reporting groups (Wang et al. 2011b). The photophysical characterization and Hg²⁺-binding properties of probe 29 in neutral N,N-dimethylformamide (DMF) aqueous solution were also investigated. The signal change of probe 29 was based on a specific metal ion-induced reversible ring-opening mechanism of the rhodamine spirolactam. The response of probe 29 for Hg²⁺ ions was instantaneous and reversible. Moreover, this probe is applied for in vivo imaging in rat Schwann cells to confirm that it can be used as a fluorescent probe for monitoring Hg²⁺ in living cells with satisfying results, which further demonstrates its value of practical applications in environmental and biological systems (Figure 19).

Figure 19:

Recognition mechanism of probe 29 with Hg²⁺.

Also in 2011, a novel fluorescent probe 30 based on rhodamine has been designed and synthesized by Li et al. for the detection of Hg²⁺ ions, which exhibited high sensitivity and selectivity over other metal ions (K⁺, Na⁺, Ca²⁺, Mg²⁺, Ba²⁺, Zn²⁺, Cd²⁺, Cu²⁺, and Pb²⁺) in aqueous solution and living cells (Wang et al. 2011c). Upon the addition of increasing concentrations of Hg²⁺, a new emission band peaking at 555 nm appeared and developed, which could be ascribed to the delocalized xanthene moiety of rhodamine group. The fluorescence response of probe 30 to other metal ions under the same condition was also investigated. There was no significant spectral change of probe 30 observed. It indicated that probe 30 could recognize Hg²⁺ from other metal ions even those that exist in high concentrations.

In 2012, Zhang et al. had a significant innovation in a new fluoroionphore-ionic liquid hybrid-based strategy to improve the performance of classic fluoroionphores via a synergistic extraction effect and realize simultaneous instrument-free detection and removal of heavy metal ions (HMIs; Jin et al. 2012). Hg²⁺ was chosen as a model HMI, and a rhodamine thiospirolactam was chosen as a model fluoroionphore to construct bifunctional fluoroionphore-ionic liquid hybrid probe 31. The novel strategy provided a general platform for the highly sensitive detection and removal of various HMIs in aqueous samples and held promise for environmental and biomedical applications (Figure 20).

Figure 20:

Structure, binding mechanism, and schematics of probe 31-based bifunctional system for the removal and highly sensitive detection of HMIs via the synergistic extraction effect.

In 2013, Zhou et al. reported a novel rhodamine-pyrene-conjugated probe 32, which exhibited lower detection limit, shorter response time, and advantageous reversibility (Chu et al. 2013). On the gradual addition of Hg²⁺ to a solution of probe 32, a new fluorescence emission band centered at 594 nm appeared, which was due to the ring opening of the rhodamine thiospirolactam. Subsequently, the emission band changed with a blue shift in the maximum to 582 nm, which might be accounted for the intermolecular electron transfer between probe 32 and Hg²⁺. Upon interaction with Hg²⁺, the result showed a 1:1 stoichiometry for the Hg²⁺ complex accompanied with a weakened fluorescence resonance energy transfer (FRET) behavior. Moreover, probe 32 could be well applied in the water sample for the detection of Hg²⁺.

A novel probe 33, 3′,6′-bis(diethylamino)-2-((2,4-dimethoxybenzylidene)amino)spiro[isoindoline-1,9′-xanthene]-3-thione, was designed and synthesized in 2013 by Gao et al. (Liu et al. 2013). Probe 33 displayed highly selective and sensitive recognition of Hg²⁺. Adding mercury ions into the aqueous solution, its fluorescence intensity was enhanced significantly, whereas its color was changed from colorless to pink. Therefore, a new fluorescence method of detection of Hg²⁺ was proposed. Satisfying results were obtained when the probe was applied in detecting Hg²⁺ in tap water, river water, and soil samples (Figure 21).

Figure 21:

Recognition mechanism of probe 33 with Hg²⁺.

In 2014, Han et al. synthesized 2-carboxybenzaldehyde rhodamine B thiohydrazine, a fluorescent probe 34, to recognize Hg²⁺ in DMF/H₂O (1:9, v/v) solution with high selectivity (Han et al. 2014). Most importantly, probe 34 could be employed to monitor Hg²⁺ in living cells using fluorescent imaging technique with satisfied results.

Emrullahoglu et al. described in 2014 the design and synthesis of a molecular probe 35 based on a rhodamine/BODIPY platform that displayed differential fluorescence responses toward Hg²⁺ and Au³⁺ and demonstrated its utility in intracellular ion imaging (Karakus et al. 2014). It exhibited a dual emission mode for the detection of Au³⁺ and a single emission mode for the detection of Hg²⁺ (Figure 22).

Figure 22:

Recognition mechanism of probe 35 with Hg²⁺.

In the same year, a rhodamine-based optical probe 36 has been developed for the selective detection of Hg²⁺ in aqueous solution as well as in living cells by Guo et al. (Aydin et al. 2014). Live cell confocal imaging study demonstrates that probe 36 is also capable of imaging the presence of Hg²⁺ ions as well as its dynamic changes in live cells (Figure 23).

Figure 23:

Recognition mechanism of probe 36 with Hg²⁺.

A core-shell structured inorganic-organic hybrid nanocomposite for Hg(II) sensing and removal was designed and fabricated in 2015 by Yang, where the core was composed of superparamagnetic Fe₃O₄ and the shell consisted of molecular silica sieve MCM-41 (Jiqu and Qixia 2015). A rhodamine-derived probe 37 was grafted onto the backbone of MCM-41 through a silane coupling reagent to control its loading content. This probe functionalized core-shell structure was confirmed and characterized by X-ray diffraction (XRD) analysis, electron microscopy images, IR spectra, thermogravimetry, and N₂ adsorption/desorption isotherms. It was found that the emission of this composite increased with increasing Hg²⁺ concentrations but was immune to other metal ions, showing good selectivity and high sensitivity toward Hg²⁺ ions. A linear Stern-Volmer curve was observed with short response time. In addition, this composite possessed good Hg²⁺ removing and recycling performance (Figure 24).

Figure 24:

Design strategy for Fe₃O₄@MCM-41@R6.

He et al. focused in 2015 on the Hg²⁺-sensing behavior of rhodamine-derived probe 38 (Shen et al. 2015). The up-conversion NaYF4 nanocrystals were constructed and applied as the excitation host so that probe 38 could be lightened by the 980 nm excited up-conversion emission, aiming at better probe photostability. The efficient energy transfer between the up-conversion host and the probes was analyzed and confirmed by spectral analysis and emission decay lifetime comparison. It was found that the probe emission linearly increased with increasing Hg²⁺ where it was immune to other common metal ions, showing emission “turn-on” effect toward Hg²⁺ with good selectivity. The probe followed a simple complexation stoichiometry of 1:1 with Hg²⁺ ions (Figure 25).

Figure 25:

Recognition mechanism of probe 38 with Hg²⁺.

A new rhodamine B-based fluorescent probe 39 containing pyrene moiety was designed and synthesized by Yao in 2016, which showed a colorimetric and fluorometric sensing ability for Hg²⁺ with high selectivity over other metal ions (Rui et al. 2016). The binding analysis using Job’s plot suggested 1:1 stoichiometry for the complexes formed for Hg²⁺. Probe 39 exhibited the linear fluorescence quenching to Hg²⁺ in the range of 0.3–4.8 μm (λ_ex=365 nm) and 0.3–5.4 μm (λ_ex=515 nm), and the detection limit was 0.72 μm. In addition, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay demonstrated that probe 39 had low cytotoxicity and was successfully used to monitor intracellular Hg²⁺ levels in living cells (Figure 26).

Figure 26:

Recognition mechanism of probe 39 with Hg²⁺.