Exploring the Scope of Photo-Induced Electron Transfer-Chelation-Enhanced Fluorescence-Fluorescence Resonance Energy Transfer Processes for Recognition and Discrimination of Zn2+, Cd2+, Hg2+, and Al3+ in a Ratiometric Manner: Application to Sea Fish Analysis.

A rhodamine-based smart probe (RHES) has been developed for trace-level detection and discrimination of multiple cations, viz. Al3+, Zn2+, Cd2+, and Hg2+ in a ratiometric manner involving photo-induced electron transfer-chelation-enhanced fluorescence-fluorescence resonance energy transfer processes. The method being very fast and highly selective allows their bare eye visualization at a physiological pH. The optimized geometry and spectral properties of RHES and its cation adducts have been analyzed by time-dependent density functional theory calculations. RHES detects as low as 1.5 × 10-9 M Al3+, 1.2 × 10-9 M Zn2+, 6.7 × 10-9 M Cd2+, and 1.7 × 10-10 M Hg2+, whereas the respective association constants are 1.33 × 105 M-1, 2.11 × 104 M-1, 1.35 × 105 M-1, and 4.09 × 105 M-1. The other common ions do not interfere. The probe is useful for intracellular imaging of Zn2+, Cd2+, and Hg2+ in squamous epithelial cells. RHES is useful for the determination of the ions in sea fish and real samples.

Introduction

A smart probe that converts a highly selective molecular recognition of multiple ions into easily detectable signals is very attractive in the present scenario. The techniques to visualize bioactive and environment-relevant cations have immense importance in biomedical analysis and environmental monitoring.[1,2] The techniques like inductively coupled plasma mass/atomic emission spectroscopy, atomic absorption spectroscopy,[3] colorimetry,[4] spectrophotometry,[5−7] and voltammetry[8] generally require sophisticated expensive equipment, tedious time-consuming sample preparation procedure, and trained skilled operator. In contrast, fluorescence spectroscopy is very useful to provide instantaneous detection, visual perception, and inexpensive methodology that excludes sample pretreatment. A single probe capable to sense multiple ions is cost-effective and highly desirable for practical applications.[9] Particularly, a probe that selectively and specifically detects and discriminates elements belonging to the same group in the periodic table through the photo-induced electron transfer–chelation-enhanced fluorescence–fluorescence resonance energy transfer (PET–CHEF–FRET) processes in a ratiometric manner is highly demanding as well as difficult to achieve[10] because it rules out the adverse environmental effects like pH, polarity, probe concentration, and excitation power on the emission intensity via built-in correction to the signal ratio of emission intensities at two different wavelengths.[11−13]

Although Al is extensively used in food packaging, cookware, antiperspirants, drinking water supplies, bleached flour, deodorants, antacids, manufacturing of cars, and computers,[14−16] it may cause neurotoxicity, disorders of homeostasis, Parkinson’s disease, Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), microcytic hypochromic anemia, myopathy, and anemia.[17−22] It also inhibits plant growth by increasing the acidity of the soil.[23]

Zinc, the most biocompatible metal,[24−27] is present in all forms of life[28] and plays a vital role in numerous biological processes including brain activity, gene transcription, immune function, etc.[29] Zn2+-based compounds are used as radioprotective agents,[30] tumor photosensitizers,[31] antidiabetic insulin-mimetic,[32] antibacterial, antimicrobial, and anticancer agents.[33] It also reduces cardio- and hepatotoxicity induced by some anticancer drugs.[34] Its abnormal metabolism causes health problems like prostate cancer, delayed sexual maturation and impotence, type 2 diabetes mellitus, ALS, Wilson’s diseases, age-related macular degeneration,[35−37] AD,[38] cerebral ischemia,[39] epilepsy,[40] and acrodermatitis enteropathica.[41]

On the other hand, Cd occupies the seventh position among the top 20 hazardous substances in the priority list prepared by the Agency for Toxic Substances and Disease Registry and the US Environmental Protection Agency.[42] The World Health Organization allows a maximum of 3 ppb Cd2+ in drinking water.[43] Cd released from Ni–Cd batteries, phosphate fertilizers, pigments, and semiconducting quantum dots/rods[44] causes renal dysfunction, calcium metabolism disorders, reduced lung capacity, risk of cancer, and health disorders.[45] Thus, the determination of trace-level Cd2+ in environmental and biological samples has great significance.

Finally, it is well-known that Hg damages kidney, skin, respiratory system, central nervous system, and other organs.[46,47] It is observed in the literature that most of the reported probes relevant to these cations either detect one or dual cations.[48−50]

Moreover, very few fluorescence probes detect and discriminate Zn2+ and Cd2+. In most cases, Cd2+ interferes with Zn2+ sensing.[51a] A simple turn-on probe[51b] for the detection of both Zn2+ and Cd2+ suffers limitations such as narrow difference in emission wavelengths (λEm for Zn2+, 572 nm and λEm for Cd2+, 565 nm) and low detection limit. An amino-terpyridine-based probe[51c] detects both Zn2+ and Cd2+ with an incremental enhancement of emission intensity at 535 nm but fails to discriminate Zn2+ and Cd2+. Chen et al.[51d] have reported a turn-on fluorescence probe for Zn2+, Cd2+, and Hg2+ that also fails to discriminate them.

It is to be noted that the molecular arithmetic that converts a chemically encoded analysis (input) into an optical signal[52,53] (output) is potentially an interesting research area in modern unconventional computing system[54] that insisted us to construct a scientific logic gate using our present probe.

Rhodamine derivatives possess excellent photophysical properties, viz. high quantum yield,[55] photostability, bioavailability, excitation, and emission in the visible region,[55] and undergo an equilibrium between the nonfluorescent “spirolactam ring” and fluorescent “ring-open” forms to allow analyte sensing through “off–on” switching.[56] Herein, a smart probe is being reported that selectively detects and discriminates Al3+, Zn2+, Cd2+, and Hg2+, utilizing their significantly different emission wavelengths and eye-catching colors under ultraviolet (UV) and visible light. The ratiometric sensing mechanism involves PET–CHEF–FRET processes. The ability of RHES for imaging intracellular Zn2+, Cd2+, and Hg2+ have been demonstrated in live cells.

Results and Discussion

Colorless RHES displays a very weak emission at 397 nm (λEx, 306 nm, Scheme , Figure S1, Supporting Information). In the presence of Al3+, Zn2+, Cd2+, and Hg2+, RHES turns pink (Al3+), green (Zn2+), sky blue (Cd2+), and intense bloodred (Hg2+) (λEx, 306 nm) (Figure ). Additionally, for Al3+ and Hg2+, the solution looks pink and red in bare eye (Figure ). Interestingly, out of all the common ions tested, the emission intensity enhances for Al3+ (51-fold along with 176 nm red shift from 397 to 573 nm), Zn2+ (29-fold along with 85 nm red shift from 397 to 482 nm), Cd2+ (36-fold along with 2 nm blue shift from 397 to 395 nm), and Hg2+ (89-fold along with 180 nm red shift from 397 to 577 nm) (Figure ).

Scheme 1

Synthetic Protocols of RHES and R1

Figure 1

Colors of RHES (20 μM) in visible light (left) and under UV light (right) in the presence of Al3+, Zn2+, Cd2+, and Hg2+ (20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES)-buffered MeOH/H2O (4/1, v/v, pH 7.4)).

Figure 2

Emission (left) and absorption spectra (right) of RHES (the same media and pH mentioned above) in the presence of the common metal ions (λEx = 306 nm).

The spectroscopic properties of RHES depend on the pH of the media as it contains pH-susceptible donor sites. Moreover, the biological application of RHES demands its efficiency at a physiological pH. Hence, RHES is mixed with Al3+, Zn2+, Cd2+, and Hg2+ in different sets at different pH values (pH 3.0–12.0). The difference in emission intensities between the free RHES and its metal ion adducts is highest near the physiological pH 7.4, and hence chosen for the entire study (Figure S2, Supporting Information).

Figures and 3a demonstrate the selectivity of RHES for Al3+ (λEm = 573 nm). Figure S3 (Supporting Information) indicates no interference from the common cations. Upon a gradual addition of Al3+ (from 0.005 to 1600 μM), the colorless RHES slowly turns sky blue, whereby the emission intensity increases at 362 nm. After 25 min, the emission intensity gradually increases at 573 nm. Further, with increasing time, the emission intensity at 362 nm remains unaltered, whereas it increases at 573 nm. It is proposed that the initial coordination of phenol −O and imine N to Al3+ is responsible for the weak emission at 362 nm. Subsequent coordination by the −O donors from the carbonyl functionality of the spirolactam moiety to Al3+ leads to fluorescence enhancement at 573 nm along with the opening of the spirolactam ring resulting in pink coloration[57] (Figure S4, Supporting Information). The time-dependent color changes of the RHES–Al3+ system under a handheld UV lamp are presented in Figure S4 (Supporting Information). Figure b shows the fluorescence titration of RHES by Al3+. RHES detects as low as 1.5 × 10–9 M Al3+ (Figure S5, Supporting Information). Figure c shows the [Al3+] versus emission intensity plot at 573 nm (red balls) and 362 nm (inset, sky blue balls). The red shift of the emission band is accompanied by a 51-fold fluorescence enhancement (λEm, 573 nm; Φ, 6.6-fold enhancement). Moreover, in the presence of Al3+, 1.5-fold increase of F573/F362 is observed (Figure S6, Supporting Information). The plot of fluorescence enhancement versus [Al3+] at different wavelengths is shown in Figure S6 (Supporting Information). The UV–vis titration of RHES with Al3+ shows the appearance of two new bands at 406 and 555 nm (Figure d), indicating the interaction of RHES with Al3+. Job’s plot supports a 1:1 stoichiometry (mole ratio) between RHES and Al3+ (Figure S7, Supporting Information). The mass spectrum of the [RHES–Al3+] adduct also affirms this composition. The binding constant of RHES for Al3+ is 1.33 × 105 (Figure S8, Supporting Information).

Figure 3

(a) Emission intensity of RHES in the presence of Al3+ and the other common metal ions (i) λEx = 306 nm, λEm = 573 nm, red bar and (ii) inset λEx = 306 nm, λEm = 362 nm, sky blue bar; (b) changes in the fluorescence spectra of RHES (20 μM) upon the gradual addition of Al3+ (0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 2.0, 5.0, 10, 50, 100, 200, 500, 800, 1200, and 1600 μM) (λEx = 306 nm); (c) plot of emission intensities of RHES vs [Al3+] at 573 nm (red balls) and at 362 nm (sky blue balls, inset), λEx = 306 nm; (d) changes in the absorption spectra of RHES in the said media upon the gradual addition of Al3+ (same as (b)).

In the presence of Zn2+, RHES experiences a red shift of the emission band to bright green along with a 29-fold fluorescence enhancement (λEm, 482 nm; λEx = 306 nm; Φ, 5.02-fold enhancement, Figure a) without any interference from the other common cations (Figure S9, Supporting Information). With an increasing [Zn2+], the emission intensity at 342 nm decreases in a ratiometric manner with an isoemissive point at 419 nm (Figure S10, Supporting Information). The plot of emission intensities versus [Zn2+] at two different wavelengths is shown in Figure S11 (Supporting Information). The fluorescence titration and emission intensities versus [Zn2+] plots are shown in Figure b,c. The absence of bands at 573 nm/577 nm, responsible for bare eye pink/red color, indicates the noninvolvement of the rhodamine moiety to coordinate to Zn2+. RHES efficiently detects Zn2+ at the physiological pH of 7.4 (Figure S2, Supporting Information) and forms a 1:1 (mole ratio) complex at a low [Zn2+], whereas a 1:2 (mole ratio) complex is formed at a higher [Zn2+], as revealed from Job’s plot (Figure S12, Supporting Information) and the mass spectrum. The binding constant of 2.11 × 104 M–1 is indicative of a fairly strong interaction between RHES and Zn2+ (Figure S13, Supporting Information). The UV–vis titration of RHES versus Zn2+ is presented in Figure d. RHES can detect Zn2+ as low as 1.2 × 10–9 M (Figure c, inset).

Figure 4

(a) Emission intensity of RHES (20 μM) in the presence of Zn2+ and the other common metal ions (λEx = 306 nm, λEm = 482 nm, olive green bar); (b) fluorescence spectra of RHES (20 mM HEPES-buffered MeOH/H2O, 4/1, v/v, pH 7.4) with increasing [Zn2+] (0.0, 0.05, 0.1, 1.0, 2.0, 5.0, 10, 20, 30, 50, 75, 100, 200, 300, 500, 1000, and 1600 μM); (c) plot of emission intensities of RHES with added Zn2+ (0.05–1600 μM) at 482 nm (red balls); (d) changes in the absorption spectra of RHES with increasing [Zn2+] (same as (b)).

In the presence of Cd2+, RHES shows a sky blue fluorescence (λEm = 395 nm, λEx = 306 nm, Figure a) without any interference from the common cations (Figure S14, Supporting Information). A gradual addition of Cd2+ enhances the emission intensity at 395 nm, while it decreases at 347 nm in a ratiometric manner with an isoemissive point at 368 nm (Figure S15, Supporting Information). The plots of emission intensities versus [Cd2+] at two different wavelengths are shown in Figure S16 (Supporting Information). The fluorescence titration with Cd2+ (0.05–1600 μM) and concentration versus fluorescence intensity are shown in Figure b,c. The absence of the fluorescence bands at 573 nm/577 nm at fluorescence titration and at 555 nm at UV titration of RHES with Cd2+ also confirm that there is no coordination with the rhodamine ring (Figure d). At a physiological pH (pH = 7.4), RHES is also efficient to detect Cd2+ (Figure S2, Supporting Information). RHES forms a 1:1 complex with Cd2+ at a low Cd2+ concentration and a 1:2 at a higher concentration, which is confirmed from Job’s plot (Figure S17, Supporting Information) and the mass spectra of the RHES–Zn2+ complex. The blue shift of the emission band is accompanied by a 36-fold fluorescence enhancement for Cd2+ at its emission point (λEm, 395 nm; Φ, 4.3-fold enhancement). The binding constant between RHES and Cd2+ is also high enough to form a stable complex (1.35 × 105 M–1) (Figure S18, Supporting Information). RHES can detect Cd2+ as low as 6.27 × 10–9 M (Figure c, inset).

Figure 5

(a) Fluorescence intensity of RHES in the presence of Cd2+ and other metal ions (λEx = 306 nm, λEm = 395 nm, blue bar); (b) fluorescence spectra of RHES (20 μM) in 20 mM HEPES-buffered MeOH/H2O (4/1, v/v, pH 7.4) upon the addition of increasing amounts of Cd2+ (0.0, 0.05, 0.1, 0.5, 1.0, 5.0, 10, 20, 50, 75, 100, 200, 300, 500, 1000, 1200, and 1600 μM) (λEx = 306 nm); (c) plot of the emission intensities of RHES (20 μM) as a function of externally added Cd2+ (0.05–1600 μM) at 395 nm (red balls); (d) changes in the absorption spectra of RHES (20 μM) in 20 mM HEPES-buffered MeOH/H2O (4/1, v/v, pH 7.4) upon the gradual addition of Cd2+ (same as (b)).

In the presence of Hg2+, the emission band of RHES undergoes a red shift with an 89-fold fluorescence enhancement (λEm, 577 nm; λEx = 306 nm; Φ, 6.5-fold increase; Figure a while its solution color turns intense bloodred under UV light and bare eye (Figure ). Upon a gradual addition of Hg2+, the emission intensity at 375 nm decreases in a ratiometric manner with an isoemissive point at 526 nm (Figure S19, Supporting Information). The plot of emission intensity versus [Hg2+] at two different wavelengths is shown in Figure S20 (Supporting Information). The fluorescence titration spectra and the plot of emission intensity versus [Hg2+] are shown in Figure b,c. The UV–vis titration of RHES versus [Hg2+] is presented in Figure d. The optimum pH for the entire study has been determined from Figure S2 (Supporting Information) as 7.4. Figure S21 (Supporting Information) reveals that the common cations do not interfere with the determination of Hg2+. Similar to Al3+, Hg2+ also forms a 1:1 (mole ratio) adduct with RHES, realized from Job’s plot (Figure S22, Supporting Information) and supported by the mass spectrum. The strong interaction between RHES and Hg2+ is indicated from the binding constant, 4.09 × 105 M–1 (Figure S23, Supporting Information). RHES detects as low as 1.7 × 10–10 M Hg2+ (Figure c, inset).

Figure 6

(a) Emission intensity of RHES (20 μM) in the presence of Hg2+ and other common metal ions (λEx = 306 nm, λEm = 577 nm, red bar); (b) changes in the emission spectra of RHES with increasing [Hg2+] (0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 2.0, 5.0, 10, 50, 100, 200, 500, 800, 1000, 1200, 1400, and 1600 μM); (c) plot of emission intensities of RHES as a function of added Hg2+ (0.005–1600 μM, red balls); (d) changes in the absorption spectra of RHES with increasing [Hg2+] (same concentration as (b), along with some more such as 20, 30, 75, 150, and 250 μM) (same media as mentioned earlier).

As the literature suggests, probably the present study is the first report of a single sensor that detects and discriminates Al3+, Zn2+, Cd2+, and Hg2+ through the generation of different colors upon irradiation of UV light and also observable in bare eye (Figure S24, Supporting Information).

Al3+ shifts the emission maxima of RHES from 397 nm (characteristic of PET, from the imine N-to the conjugated 3-ethoxysalicylaldehyde moiety) to 573 nm (characteristic of rhodamine B, FRET) via an intermediate CHEF process having a characteristic emission at 362 nm. Al3+ being a hard acid prefers an “O” donor from 3-ethoxysalicylaldehyde. The absorption at 406 nm also indicates the interaction between Al3+ and the “O” donor from 3-ethoxysalicylaldehyde (Figure d). Thus, upon excitation at 306 nm, the fluorescence at 362 nm enhances because of the CHEF process which subsequently transfers energy to excite the rhodamine moiety.

However, Hg2+ being a soft acid does not prefer the hard “O” donor, and hence the CHEF process is absent. Rather, a significant overlap between 3-ethoxysalicylaldehyde emission (donor) and rhodamine absorption (acceptor) has been observed (Figure S25, Supporting Information). Upon the addition of Hg2+, the emission maximum of free RHES is red-shifted from 397 to 577 nm (λEx, 306 nm), indicating an energy transfer from the donor to the acceptor.

The slow interaction between RHES and Al3+ allows the intermediate CHEF process visible. With time, the intensity at 573 nm gradually increases, and finally it moves over to 362 nm. Hence, an intense pink fluorescence and bare eye pink coloration are observed (Figure S26, Supporting Information). The pseudo-first-order rate constant for the interaction has been estimated as 0.0413 min–1 by monitoring the changes in the emission intensity at 573 nm (Figure S27, Supporting Information). The proposed binding mechanism is further substantiated by a control experiment using a model compound R1 that lacks −OH functionality. In the presence of Al3+/Hg2+, the emission spectra of R1 are significantly different (Figures S28, S29, Supporting Information) while their QTOF–MS spectra support the formation of the adduct (Figures S30, S31, Supporting Information). On the basis of these facts, a probable interaction mechanism of RHES with Al3+, Zn2+, Cd2+, and Hg2+ is shown in Scheme .

Scheme 2

Proposed Binding Mechanism

As far as the emission profile is concerned (Figure S32, Supporting Information), Zn2+/Cd2+ do not show any significant interaction with R1, suggesting the necessity and involvement of the phenol −OH for their sensing. For a deeper understanding of the binding mode of RHES and to strengthen the proposed sensing mechanism, 1H NMR titration has been conducted in dimethyl sulfoxide (DMSO)-d6. After the addition of 1 equiv Al3+/Hg2+ to RHES, the alkane protons downfield-shifted from 3.45 to 3.50 ppm, indicating the interaction of the adjacent O donors with the cations. Upon the addition of more Al3+/Hg2+ (2.0 and 3.0 equiv), these protons further downfield-shifted to 3.54 ppm. Moreover, the ring protons upfield-shifted from 7.0 to 6.95 ppm, which indicates the opening of the spirolactam ring. Interestingly, after the addition of 1 equiv Al3+/Hg2+ to RHES, the “p” proton downfield-shifted from 8.6 to 10.2 ppm (Al3+) and 10.3 ppm (Hg2+) because of the close proximity of the rhodamine B unit through the Al3+/Hg2+-induced folding of RHES. A further addition of Al3+/Hg2+ (2.0 and 3.0 equiv) shifts the “p” proton more downfield. The position of the “v” proton of RHES remains unchanged upon the addition of Hg2+, indicating the noninteraction of the −OH group with Hg2+, whereas it shifts downfield from 12.01 to 12.44 for Al3+, confirming the interaction of the −OH group with Al3+ (Figures S33, S34, Supporting Information).

Similarly, the addition of 1 equiv of Zn2+/Cd2+ to RHES shifts the alkane protons downfield from 3.5 to 3.6 ppm, indicating the interaction of the adjacent O donors to the metal ions. These protons are further downfield-shifted upon the addition of more Zn2+/Cd2+ (2.0 and 3.0 equiv). Moreover, the CH=N proton (“p” proton) is downfield-shifted from 8.75 to 10.25 ppm, whereas the “v” proton shifts downfield from 12.0 to 12.37, indicating the involvement of the −OH group to Zn2+/Cd2+ binding (Figures S35, 36, Supporting Information).

To further support the sensing mechanism, fluorescence lifetime measurement is carried out. The average lifetime (τ) of RHES is 0.3852 ns, whereas the corresponding values for RHES–Al3+ (λEm = 573 nm) and RHES–Hg2+ (λEm = 577 nm) systems are 1.7497 and 1.7257 ns, respectively. The τ values for the RHES–Zn2+ (λEm = 482 nm) and RHES–Cd2+ (λEm = 395 nm) systems are 1.2791 and 1.5333 ns, respectively (Table S1, Supporting Information). The τ value of [RHES–Al3+] at 362 nm is 0.8399 ns (Table S1, Figure S37, Supporting Information). The fluorescence lifetime decay curves along with data fitting are shown in Figure .

Figure 7

Fluorescence lifetime decay of RHES and (a) [RHES–Al3+], (b) [RHES–Zn2+], (c) [RHES–Hg2+], and (d) [RHES–Cd2+] adducts at the corresponding emission wavelengths.

Interestingly, the higher affinity and association constant of RHES for Cd2+ allows easy replacement of Zn2+ from the [RHES–Zn2+] adduct to form a more stable [RHES–Cd2+] adduct (Scheme ). Thus, the [RHES–Zn2+] adduct turns out to be a better Cd2+ sensor that functions via the displacement approach. The gradual addition of Cd2+ to the [RHES–Zn2+] system results in a blue shift of the 482 nm emission band to 395 nm along with a fluorescence enhancement (Figure ).

Scheme 3

Easy Replacement of Zn2+ by Cd2+ from the [RHES–Zn2+] Adduct

Figure 8

(a) Changes in the fluorescence spectra of the [RHES–Zn2+] adduct upon the addition of Cd2+; (b) changes in the absorbance spectra of the [RHES–Zn2+] adduct upon the addition of Cd2+.

It is worth mentioning that the simultaneous presence of Al3+ and Hg2+ can be visualized (Scheme ) using KI to mask Hg2+, while KI does not interfere with the emission profile of RHES (Figure S38, Supporting Information). This fact is also supported from the mass spectrum of the resulting mixture (Figure S39, Supporting Information). Figure S40 (Supporting Information) pictorially represents the reversibility of the probe toward Hg2+ detection. After the addition of the mixture of Al3+ and Hg2+ to RHES, the characteristic emission of the [RHES–Hg2+] adduct at 577 nm is observed, attributed to the higher affinity of Hg2+ for RHES. The higher binding constant of [RHES–Hg2+] over [RHES–Al3+] also supports the observation (Figures S8, S23, Supporting Information). Figure S41 (Supporting Information) also indicates the formation of the [RHES–Hg2+] adduct (m/z, 1007.86) when the mixture of Al3+ and Hg2+ is added to RHES. Moreover, after the addition of I– to this system, the emission shifts to 573 nm, which is characteristic of the [RHES–Al3+] adduct. After the addition of I–, it captures Hg2+ while only Al3+ binds to RHES, showing an emission at 573 nm. The I–-assisted reversible interaction between the probe RHES and Hg2+ is also established from the 1H NMR titration (Figure S42, Supporting Information). Upon the addition of 1 equiv Hg2+ to the [RHES–Al3+] adduct, the “V” proton of RHES upfield-shifted from 12.34 to 12.15 ppm, indicating the noninteraction of the −OH group with Hg2+ and the replacement of Al3+ by Hg2+. Upon further addition of I– (1 equiv) to the resulting solution, the “V” proton is downfield-shifted from 12.15 to 12.34 ppm, indicating the interaction of the −OH group of RHES. This is due to the interaction of Al3+ present in the system with the free RHES. The formation of this [RHES–Al3+] adduct is also demonstrated from its Fourier transform infrared (FTIR) spectrum (Figure S43, Supporting Information).

Scheme 4

Role of the Masking Agent (KI) for Simultaneous Detection of Al3+ and Hg2+

Table S2 (Supporting Information) compares the present probe with the available probes in the literature, although it is not truly a comparison because none of the available single probes can detect all the four cations simultaneously as demonstrated in the present report.

To obtain the energy-optimized geometries of RHES and its Al3+, Hg2+, Zn2+, and Cd2+ adducts (Figure S44, Supporting Information), density functional theoretical (DFT) calculations[57] have been performed. The Gaussian-09 revision C.01 program package is used for all calculations. The geometries in gas phase are fully optimized without any restrictions of symmetry in singlet ground states for RHES and its adducts, viz. [Al(RHES)(NO3)3], [Zn(RHES)(CH3CH2OH)2(CH3OH)(NO3)]+, [Cd(RHES)(CH3CH2OH)(CH3COO)], and [Hg(RHES)(NO3)]+, along with the gradient-corrected DFT level with the three-parameter fit of exchange and correlation functional of Becke (B3LYP), which includes the correlation functional of Lee, Yang and Parr (LYP).[57b] The basis set LanL2DZ along with an effective core potential is employed for Al, Zn, Cd, and Hg atoms following the associated valence double-ζ basis set of Hay and Wadt[57c] along with the 6-31++G** basis set, chosen for hydrogen, carbon, nitrogen and oxygen. The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of RHES and its adducts are shown in Figure S45 (Supporting Information). It is clear that the Al3+, Hg2+, Zn2+, and Cd2+ adducts are stabilized by 0.06227, 0.03886, 0.0721, and 0.07823 eV, respectively. In [RHES–Al3+] and [RHES–Hg2+], the HOMOs are found to be majorly localized on the rhodamine moiety, whereas these are localized on the aldehyde moiety in the case of the [RHES–Zn2+] and [RHES–Cd2+] adducts. The corresponding LUMOs are found to be minorly localized on the rhodamine moiety. Time-dependent DFT (TDDFT) calculations have been performed on the excited state based on the optimized ground-state geometry in the Conductor-like Polarizable Continuum Model (CPCM). The electronic transition energies are calculated by the TDDFT method in methanol and gas phase (Tables S3–S7, Supporting Information). The outcomes are in good agreement with our experimental data. The results reveal that the energy gap between HOMO and LUMO of its adducts is reasonably lower than that of the free RHES, leading to a more stable species.

Considering the emission wavelengths of Zn2+ and Cd2+ and the displacement of Zn2+ by Cd2+, a binary logic gate has been constructed.[58] There are two input signals, viz. input X (Zn2+) and input Y (Cd2+), whereas the output signals correspond to the turn-on emission at 482 nm for Zn2+ and 395 nm for Cd2+. For input, Zn2+/Cd2+ is defined as the “1” state and absence of any as the “0” state. Our system uses the OR gate and the combination of OR and NOT gates along with a switch that describes the situation clearly. When the input is applied through an individual, both ions follow the OR gate, and when both Zn2+/Cd2+ are the inputs, the pathway is changed (Figure ).

Figure 9

Truth table diagram for simultaneous monitoring of Zn2+ and Cd2+ as inputs, attributed to the enhancement of emission intensity as the output.

Imaging Studies

Imaging System

The imaging system is composed of an inverted fluorescence microscope Leica DM 1000 LED, a digital compact camera Leica DFC 420C, and an image processor Leica Application Suite v3.3.0. The microscope is equipped with a 50 W mercury arc lamp in a 25 min interval after incubation with the said ions to RHES.

Staining with RHES

The slides are deparaffinized in xylene, hydrated through a graded series of ethanol, equilibrated in 1 mL Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) at 37 °C with 5% CO2, incubated with RHES for 2 min, washed several times with 2% DMSO, and observed under a fluorescence microscope.

Image Analysis

RHES efficiently images Zn2+, Cd2+, and Hg2+ in squamous epithelial cells under a fluorescence microscope (Figure ). The cells are cultured in a 35 mm culture plate at a seeding density of 40 × 104 cell/35 mm in 1 mL DMEM with 10% FBS at 37 °C with 5% CO2. To explore the suitability of RHES for the bioimaging of Zn2+, Cd2+, and Hg2+, the cells are incubated in the presence or absence of 20 mg mL–1 of RHES for 2 h at 37 °C and 5% CO2 in a culture medium. Before this, the cells are subcultured using 0.25% trypsin–0.53 mM ethylenediaminetetraacetic acid solution in a 60 mm plate. The cells are washed with phosphate-buffered saline (PBS) (×2), and after washing with PBS (×3), the remaining compounds are removed, and the cells are incubated with the said ions and observed under a fluorescence microscope equipped with a UV filter at ambient temperature. No fluorescence is observed in the cells (Figure , RHES) that are not previously exposed to the metal ions, whereas after the addition of the said ions, the nonfluorescence gradually turns green with time for Zn2+, sky blue for Cd2+, and red for Hg2+. Moreover, RHES can easily permeate through the tested living cells without causing any harm (as the cells remain alive even after 2 h of exposure to RHES). The cells treated only with the said ions have no fluorescence. This intracellular imaging clearly shows that RHES has good cell permeability and is efficient for imaging of the said cations.

Figure 10

Fluorescence microscopic images of squamous epithelial cells after incubation for 2 h (100× objective lens): (a) RHES (20 μM); (b) RHES (20 μM) + Zn2+ (30 μM); (c) RHES (20 μM) + Cd2+ (30 μM); (d) RHES (20 μM) + Hg2+ (30 μM).

Application

Real Sample Analysis

To evaluate the practical feasibility of RHES for the determination of Al3+, Zn2+, Cd2+, and Hg2+, analyses of the real samples have been performed.[9,58] The results are summarized in Tables –4, respectively. Table indicates the excellent recovery of metal ions from both water and the antacid samples, opening a new avenue for the determination of Al3+ in pharmaceutical formulations and water samples. The method has also been employed successfully for the determination of Zn2+ in water and commercially available antacid-suspension supplements (Table ). Tables and 4 indicate the determination of Hg2+ and Cd2+ ions in sea fish samples, respectively. The results indicate excellent recoveries of the said cations from the real samples.

Table 1

Determination of Al3+ in Real Samples

sample	Al3+ added (μM)	emission intensity (au)	Al3+ found (μM)	RSD (%)	recovery (%)
drinking water	10	423	9.89	1.7	98
industrial water		451	10.7	1.8
antacid-suspension	12	561	11.71	1.7	97

Table 4

Determination of Cd2+ in Sea Fish Samples

sample no.	fish name	dry weight of the samples	Cd2+ (μg/g) present	emission intensity (au)	RDS (%)
S1	Subgenus Thunnus	1.375	0.381	51	1.7
S2	Eleutheronema tetradactylum	0.237
S3	Loligo duvauceli	1.323	0.176	44	1.7
S4	Setipinna sp.	0.273	0.094	21	1.8
S5	Johnieops vogleri	1.216	0.264	32	1.5
S6	Stolephorus indicus	0.579	0.291	40	1.7
S7	Pampus argenteus	0.827	0.182	28	1.9
S8	Parupeneus indicus	0.557	0.161	31	1.7

Table 3

Determination of Zn2+ in Real Samples

sample no.	Zn2+ added (μM)	emission intensity (au)	Zn2+ found (μM)	RDS (%)	recovery (%)
drinking water	13	210	12.89	1.7	99
industrial water		269	11.32	1.9
antacid-suspension	15	240	14.76	1.9	97

Table 2

Determination of Hg2+ in Sea Fish Samples

sample no.	fish name	dry weight of the samples	Hg2+ (μg/g) present	emission intensity (au)	RDS (%)
S1	Subgenus Thunnus	1.285	1.81	97	1.7
S2	Eleutheronema tetradactylum	0.326
S3	Loligo duvauceli	1.321	0.149	43	1.5
S4	Setipinna sp.	0.3536	0.291	51	1.8
S5	Johnieops vogleri	1.21	0.11	35	1.7
S6	Stolephorus indicus	0.571	0.239	56	1.7
S7	Pampus argenteus	0.826	0.381	67	1.6
S8	Parupeneus indicus	0.467	0.167	41	1.9

Fish Assays

Collection and Storage

The fish samples are collected from the coastal areas of West Bengal (Digha), India, during January, 2016. Eight fishes, namely subgenus Thunnus, Eleutheronema tetradactylum, Loligoduvauceli, Setipinna sp, Johnieopsvogleri, Stolephorusindicus, Pampus argenteus, and Parupeneusindicus are collected from the coastal areas. The body weight and length of the fishes are measured prior to dissection. The muscle sample is collected and stored in sterile polythene bags and kept in the laboratory deep freezer (−20 °C) to prevent deterioration till further analysis.

Analysis

The developed method is used to determine Cd2+ and Hg2+ ions quantitatively in sea fish samples. To obtain the samples in the form of dry powder, the fishes are lyophilized using a Virtis freeze mobile 6EL lyophilizer (UGC-DAE CSR, Kolkata). As the accumulated Cd2+ and Hg2+ in the fish samples are in covalent form, they are not suitable for fluorescence measurements. Hence, the fish samples are digested in a minimum volume of concentrated HNO3 to obtain Cd2+ and Hg2+ in salt form. The sample preparation and analysis procedures are presented in the Supporting Information. The concentrations of Cd2+ and Hg2+ are measured by the standard addition method using the linear plot, as in Figure S46 (Supporting Information). The emission intensity is measured at pH 7.4.

Recovery Studies of Zn2+ and Al3+

RHES is also used for the determination of Al3+ and Zn2+ in real samples following the standard addition method described above through spiking the known amounts of these two metal ions at different concentration levels. The results are summarized in Tables and 3. The sample preparation and analysis procedures are presented in the Supporting Information. Using the calibration graph (Figure S47, Supporting Information), the amounts of unknown Al3+ and Zn2+ have been determined.

Conclusion

The synthesis, characterization, and application of a very simple fluorescence and colorimetric probe, RHES, is described. RHES is exploited for the detection of nanomolar Al3+, Zn2+, Cd2+, and Hg2+ ions in a ratiometric manner involving the PET–CHEF–FRET processes. The method allows their bare eye visualization at a physiological pH. It detects as low as 1.5 × 10–9 M Al3+, 1.2 × 10–9 M Zn2+, 6.7 × 10–9 M Cd2+, and 1.7 × 10–10 M Hg2+. Furthermore, RHES successfully images the cations that belong to the same group of the modern periodic table, viz. Zn2+, Cd2+, and Hg2+, in living cells. The developed method is useful for the determination of the said ions in real and sea fish samples. RHES, a multi-ion sensor, has been used to construct a binary logic gate. The DFT studies support the experimental findings.

Experimental Section

Materials and Methods

High-purity HEPES buffer, rhodamine B, 3-ethoxysalicylaldehyde, 4-formylbenzonitrile, and 2-[2-(2-amino-ethoxy)-ethoxy]-ethylamine have been purchased from Sigma-Aldrich (India). Hg(NO3)2·6H2O, Al(NO3)3·6H2O, Zn(NO3)2·6H2O, and Cd(AcO)2·4H2O are purchased from Merck (India). Spectroscopic grade solvents are used. All metal salts used are of either nitrate or acetate form. The other chemicals are of analytical reagent grade and used without further purification. Ultrapure Milli-Q Millipore 18.2 MΩ cm–1 water is used whenever required. A Shimadzu Multi Spec 2450 spectrophotometer is used for recording the UV–vis spectra. The FTIR spectra are recorded on a Shimadzu FTIR (model IRPrestige 21 CE) spectrophotometer. The mass spectra are recorded with a QTOF 60 Micro YA 263 mass spectrometer in ES-positive mode. The 1H NMR and 13C NMR spectra are measured with Bruker ADVANCE 400 (400 MHz) and 300 (75 MHz) spectrometers. Time-resolved fluorescence lifetime measurements are performed with a picosecond-pulsed diode laser-based time-correlated single-photon counting spectrometer (IBH, UK, λex = 380 nm) coupled to an MCP-PMT detector. The data are fitted to multiexponential functions after the deconvolution of the instrument response function by an iterative reconvolution technique using the IBH DAS 6.2 data analysis software. The steady-state emission and excitation spectra are recorded with a Hitachi F-4500 spectrofluorimeter. A digital SYSTRONICS pH meter (model 335) is used for pH measurement.

Synthesis of RHE

2-[2-(2-Amino-ethoxy)-ethoxy]-ethylamine (7.0 mL, 12.42 mmol, ρ = 1.015 g mL–1) is added dropwise to 1.1 g rhodamine B (2.29 mmol) in methanol (30 mL) under stirring condition at room temperature. The mixture is refluxed for 4 days at 60 °C (Scheme ). The solvent is removed under reduced pressure using a rotary evaporator. Then, HCl (1 mol L–1) is added until the solution becomes clear. The pH of the solution is adjusted to 9–10 using NaOH (1 mol L–1). A red precipitate that appeared is filtered, washed with water, dried under a vacuum, and assigned as RHE, 2-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3′,6′-bis(diethylamino)spiro[isoindoline-1,9′-xanthen]-3-one (yield: 95.8%). Anal. Calcd (%): C, 71.31; H, 7.74; and N, 9.78. Found: C, 72.05; H, 7.92; and N, 9.25. QTOF–MS ES+ (Figure S48, Supporting Information): [M + H]+ = 573.36 (∼100%). FTIR (cm–1) (Figure S49, Supporting Information): ν(N–H, 1° amine) 3289.23; ν(C–H, aromatic) 2967.21, 2869.56; ν(C=O, carbonyl, rhodamine spirolactum ring) 1673.81; ν(C=C, aromatic) 1515.22; ν(C=C) 1456.23; ν(C–N, carbonyl group) 1389.65; ν(C–O, xanthan ring) 1232.10, 1098.78.

Synthesis of RHES

The probe, RHES, has been synthesized by refluxing the equimolar mixture of RHE (1.03 g, 1.80 mmol) and 3-ethoxysalicylaldehyde (0.30 g, 1.80 mmol) in methanol for 6 h at 60 °C (Scheme ). The red gel obtained after the evaporation of the solvent is assigned as RHES, (E)-3′,6′-bis(diethylamino)-2-(2-(2-(2-((2-hydroxy-3-(λ3-oxidanyl)benzylidene)amino)ethoxy)ethoxy)ethyl)spiro[isoindoline-1,9′-xanthen]-3-one (0.60 g, yield: 96%). Anal. Calcd (%): C, 71.64; H, 7.27; and N, 7.77. Found: C, 71.95; H, 7.37; and N, 7.02. QTOF–MS ES+ (Figure S50, Supporting Information): [M + H]+ = 721.93 (∼40%) and [M + Na]+ = 743.91 (∼100%). 1H NMR (Figure S51, Supporting Information) (400 MHz, CDCl3): δ (ppm) 13.163 (1H, s), 8.290 (1H, s), 7.895 (5H, m, J = 3.2), 7.433 (5H, m, J = 4.8), 7.062–6.719 (2H, m, J = 2.8), and 6.430–6.228 (4H, m, J = 4.4, xanthene moiety). 13C NMR (Figure S52, Supporting Information) (100 MHz, CDCl3): δ (ppm) 168.39, 166.64, 153.8, 153.34, 152.57, 148.91, 148.81, 132.47, 131.06, 129.03, 128.94, 128.05, 123.87, 123.27, 122.85, 117.78, 115.63, 108.17, 105.61, 97.88, 97.97, 77.47, 77.15, 76.84, 70.62, 69.96, 67.95, 64.96, 58.55, 44.46, and 39.40. FTIR (cm–1) (Figure S53, Supporting Information): ν(O–H) 3373.50; ν(C–H, aromatic) 2974.23, 2904.80; ν(CH=N, imine bond) 1678.07; ν(C=C, stretch) 1618.28; ν(C=O, carbonyl) 1512.19; ν(C–N, stretch) 1381.03; and ν(C–O, stretch) 1232.51, 1068.56. The absorption spectrum (MeOH/H2O, 4/1, v/v, 20 mM HEPES, pH 7.4, Figure S54, Supporting Information) shows two peaks, viz. λ (ε, M–1 cm–1) 273 nm (6.3 × 104), assigned to the π–π* transition, and 312 nm (5.93 × 102), assigned to the n−π* transition (from the nonbonding terminal N of the xanthane moiety to π* of RHES). RHES emits at 397 nm (λEx, 306 nm, same media, Figure S52, Supporting Information).

Synthesis of R1

The model probe R1 has been synthesized by refluxing an equimolar mixture of RHE (0.50 g, 0.87 mmol) and 4-formylbenzonitrile (0.11 g, 0.87 mmol) in methanol for 6 h at 60 °C (Scheme ). The red gel obtained after evaporation of the solvent is assigned as R1, (E)-4-(((2-(2-(2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)ethoxy)ethoxy)ethyl)imino)methyl)benzonitrile (0.108 g, yield, 95%). Anal. Calcd (%): C, 73.64; H, 6.91; and N, 10.21. Found: C, 74.05; H, 7.07; and N, 9.97. QTOF–MS ES+ (Figure S55, Supporting Information): [M + H]+ = 685.6366 (∼100%). FTIR (cm–1) (Figure S56, Supporting Information): ν(C–H, aromatic) 2968.45; ν(C≡N) 2362.80; ν(C=C, stretch) 1664.57 and 1610.56; ν(C=O, carbonyl) 1512.19; ν(C–N, stretch) 1381.03; ν(C–O, stretch) 1265.30, 1222.87, and 1114.86.

Synthesis of the [RHES–Al3+] Adduct

The methanol solution of Al(NO3)3·6H2O (0.3 g, 0.8 mmol) is added dropwise to a magnetically stirred solution of RHES (0.57 g, 0.8 mmol) in methanol at room temperature. Upon slow evaporation of the solvent, an intense pink color solid has been obtained. The yield is 80%. Anal. Calcd (%): C, 55.30; H, 5.61; N, 10.50; and Al, 2.89. Found: C, 55.98; H, 5.50; and N, 11.40. QTOF–MS ES+ (Figure S57, Supporting Information): m/z, 956.83 (∼100%) is assigned to [RHES + Al3+ + 3NO3– + Na]+, indicating a 1:1 (mole ratio) stoichiometry between RHES and Al3+. FTIR (cm–1) (Figure S58, Supporting Information): ν(C–H, stretch) 2995.29; ν(C–O, carbonyl) 1618.28; ν(N–O) 1390.68; and ν(C–O) 1305.81. UV–vis (Figure S59, Supporting Information): λ (nm) in MeOH/H2O, 4/1, v/v (ε, M–1 cm–1): 406 nm (5.6 × 102), 555 nm (7.5 × 106). The excitation spectrum (λEm, 573 nm) is presented in Figure S59 (Supporting Information).

Synthesis of the [RHES–Zn2+] Adduct

The methanol solution of Zn(NO3)2·6H2O (0.3 g, 1.01 mmol) is added dropwise to a magnetically stirred solution of RHES (0.73 g, 1.01 mmol) in methanol at room temperature. Upon the removal of the solvent by slow evaporation, a light yellow solid appears, which is assigned as the [RHES–Zn2+] adduct (yield, 85%). Anal. Calcd (%): C, 59.53; H, 6.95; and N, 7.21. Found: C, 59.12; H, 6.75; and N, 7.21. QTOF–MS ES+ (Figure S60, Supporting Information): m/z, 992.03 is assigned to [RHES + Zn2+ + 2CH3CH2OH + CH3OH + H]+ (∼100%), indicating a 1:1 (mole ratio) stoichiometry between RHES and Zn2+, whereas m/z, 1123.04 is assigned to [RHES + 2Zn2+ + 2CH3CH2OH + 2NO3– + 2H2O + H]+ (∼40%), indicating a 1:2 (mole ratio) stoichiometry between RHES and Zn2+. FTIR (cm–1) (Figure S61, Supporting Information): ν(C–H) 2970.38; ν(C=O, carbonyl) 1739.19; ν(C=N, imine bond) 1614.42; ν(C=C, aromatic) 1377.17; ν(C–N, attached with the carbonyl group) 137.39; and (C–O, stretch) 1217.08. UV–vis (Figure S62, Supporting Information): λ (nm) in MeOH/H2O, 4/1, v/v (ε, M–1 cm–1): 314 nm (5.6 × 102), 273 nm (7.5 × 106). The excitation spectrum (λEm, 482 nm) is presented in Figure S62 (Supporting Information).

Synthesis of the [RHES–Cd2+] Adduct

The methanol solution of Cd(NO3)2·4H2O (0.3 g, 0.7 mmol) is added dropwise to a magnetically stirred solution of RHES (0.38 g, 0.7 mmol) in methanol at room temperature. A slow evaporation of the solvent yielded a gray solid, ascribed as the [RHES–Cd2+] adduct (yield, 82%). Anal. Calcd (%): C, 52.36; H, 5.97; and N, 4.20. Found: C, 52.36; H, 5.89; and N, 4.29. QTOF–MS ES+ (Figure S63, Supporting Information): m/z, 1020.15 is assigned to [RHES + Cd2+ + 2CH3COO– + CH3CH2OH + H]+ (∼100%), indicating a 1:1 (mole ratio) stoichiometry between RHES and Cd2+, whereas m/z, 1170.21 is assigned to [RHES + 2Cd2+ + 2CH3COO– + CH3O– + 3H2O + H]+ (∼50%), confirming a 1:2 (mole ratio) stoichiometry between RHES and Cd2+. FTIR (cm–1) (Figure S64, Supporting Information): ν(C–H) 2970.38; ν(C=O, carbonyl) 1737.86; and ν(C–O) 1217.08. UV–vis (Figure S18, Supporting Information): λ (nm) in MeOH/H2O, 4/1, v/v (ε, M–1 cm–1): 273 nm (5.6 × 106), 311 nm (7.5 × 102). The excitation spectrum (λEm, 395 nm) is presented in Figure S65 (Supporting Information).

Synthesis of the [RHES–Hg2+] Adduct

The methanol solution of Hg(NO3)2·6H2O (0.3 g, 0.7 mmol) is added dropwise to a magnetically stirred solution of RHES (0.49 g, 0.7 mmol) in methanol at room temperature. A slow evaporation of the solvent produced bloodred solid (yield, 91%). Anal. Calcd (%): C, 52.51; H, 5.33; and N, 7.12. Found: C, 52.93; H, 5.12; and N, 7.02. QTOF–MS ES+ (Figure S66, Supporting Information): m/z, 1007.77 is assigned to [RHES + Hg2+ + NO3– + Na]+ (∼100%), indicating a 1:1 (mole ratio) stoichiometry between RHES and Hg2+. FTIR (cm–1) (Figure S67, Supporting Information): ν(C–H) 2980.02; ν(C–O, carbonyl) 1672.28; ν(C–N) 1377.17; and ν(C–O, attached with the carbonyl group) 1303.88, 1072.42. UV–vis (Figure S68, Supporting Information): λ (nm) in MeOH/H2O, 4/1, v/v (ε, M–1 cm–1): 555 nm (5.6 × 106). The excitation spectrum (λEm, 577 nm) is presented in Figure S68 (Supporting Information).