A Three Dimensional (Time, Wavelength and Intensity) Functioning Fluorescent Probe for the Selective Recognition/Discrimination of Cu 2+ , Hg 2+ , Fe 3+ and F - ions

W E HAVE STRATEGICALLY INCORPORATED THREE DIFFERENT FLUOROPHORES AT TREN TO CONSTRUCT A MULTI - ENERGY DONOR / ACCEPTOR “ SMART ” PROBE L. T HIS PROBE OPERATES BY USING THREE DIMENSIONAL SCALES ( response time, wavelength and fluorescence intensity) which allows for the SELECTIVE RECOGNITION AND DISCRIMINATION OF THE IONS Cu 2+ , Hg 2+ , Fe 3+ and F - .

initiate three different FRET recognition modes dependent on the different target ions present, viz recognition of Hg 2+ via the Rh-B moiety (585 nm), Fe 3+ via both the Rh-B (585 nm) and NBD moieties (525 nm), whilst Cu 2+ is only through the NBD moiety (525 nm). Similar phenomenon can be observed when we tuned the excitation wavelength to 470 nm (λex(NBD)), except that the fluorescence intensity was stronger (Fig. S5). This stronger fluorescence maybe attributed to the fact that the overlap of the spectral area of NBD/Rh-B is larger than that of coumarin/Rh-B (Fig. S4), which resulted in a higher efficient FRET process.
In order to investigate these three different FRET "OFF-ON" systems, fluorescent titration experiments have been carried out. Upon increasing the concentration of Hg 2+ in a solution of probe L (Fig. 3a), the emission maximum shifted from 525 nm (characteristic of NBD, weak emission due to the PET effect) to 585 nm (characteristic of Rh-B, the acute enhancement is attributed to the Hg 2+ -induced FRET) via an intermediate chelation process with a characteristic emission band at about 555 nm (upon addition of 0.1 equiv. of Hg 2+ ). It reached equilibrium after the addition of 1.0 equiv. Hg 2+ which suggested a 1:1 complex stoichiometry for the Hg 2+ ·Rh-B-L complex (Fig. 3a  inset). Interestingly, when Fe 3+ was added to the solution of probe L, at the beginning (0 to 1.0 equiv.), there was no significant fluorescence change at 585 nm, but it did exhibit a detectable fluorescent enhancement at 525 nm which maybe ascribed to the inhibition PET process of the NBD fluorophore (Fig. 3b). On increasing the concentration of Fe 3+ from 1.0 to 2.0 equiv., a dramatically fluorescence enhancement was observed at 585 nm, which indicated the Fe 3+ -induced spirolactam ring-opening of Rh-B moiety during this process. Further increasing Fe 3+ ( >2.0 equiv.) did not bring any significant changes in the fluorescent spectrum, which suggested that the complex system had reached equilibrium. Hence, these observation indicated that there is a two-step complex process for probe L and Fe 3+ : firstly, probe L complexes with one equiv. of Fe 3+ through the NBD moiety forming an Fe 3+ ·NBD-L intermediate complex, and then further complexes Fe 3+ through the Rh-B moiety to form the final stable 1:2 Fe 3+ ·NBD-L-Rh-B·Fe 3+ complex. It is perhaps this two-step complex process which results in the longer observed response time for the detection of Fe 3+ by probe L. The significant emission at 585 nm (Rh-B) and a relatively weaker emission band at 525 nm (NBD) appeared, which suggested that two FRET processes were occurring between coumaring-Rh-B and coumaring-NBD. However, on increasing the amount of Cu 2+ cation in the solution of probe L, a green fluorescence was induced which was ascribed to the emission of the NBD fluorophore at 525 nm ( Fig. 3c, to more fully explore the complexation process, the 470 nm excitation titration results are shown.). The results strongly suggest that the probe L·Cu 2+ complex was formed through the NBD moiety, i.e. Cu 2+ ·NBD-L. The 1:1 complex stoichiometry for Cu 2+ ·L complex was supported by a Job's plot (Fig. S6).
On the other hand, in order to fully investigate the recognition capability of probe L, studies of probe L towards anions have been carried out in CH3CN solution (Fig. 4). Regardless of whether 365 nm or 470 nm were employed as the excitation wavelength, there was only a very weaker fluorescence at about 525 nm (Iλ365 nm = 30 and Iλ470 nm = 95) for probe L. Interestingly, upon the addition of Fanions, the different excitation wavelength resulted in different phenomenon. When 365 nm was employed as the excitation wavelength, the fluorescence exhibited an acute increase at 460 nm, which indicated that the coumarine participates in the complexation of F - (Fig. 4a). In contrast, when 470 nm was selected as the excitation wavelength, the fluorescence at 525 nm decreased upon the addition of Fwhich suggested that the NBD moiety also participated in the complexation process (Fig. S7). The fluorescent titration experiments revealed the detail complex process (Fig. S8), namely that the Fanion was complexed by the OH group at the coumarine moiety and the NH group at the NBD moiety via hydrogen bonding. This binding interaction locks the C=N bond of probe L in place, preventing its rapid isomerisation and switching the fluorescence 'on'.
To further investigate the practical applicability of probe L (20 M) as a Hg 2+ , Fe 3+ , Cu 2+ and Fion selective fluorescent probe, competitive experiments were carried out. As shown in Fig. S9, no significant interference to the selective response of probe L to Hg 2+ , Fe 3+ , Cu 2+ or Fion were observed in the presence of any of the other ions employed herein when using the three dimensional scales (time, wavelength and intensity) method. Upon the addition of probe L to the detection solution, if we observed an acute enhancement and stable fluorescence at 585 nm (orange coloured solution) over two minutes, this is suggestive of the presence of Hg 2+ . By contrast, if we observe a gradual enhancement (over about 100 min) at 585 nm and 525 nm, this is consistent with the presence of Fe 3+ ; on the other hand, if we only observe a fluorescence enhancement at 525 nm (a green coloured solution), this suggests the presence of Cu 2+ . Furthermore, if we observe a fluorescent enhancement at 460 nm (a cyan coloured solution), this indicates the presence of Fanion. In other words, probe L can act as a highly efficient three dimensional scale fluorescent probe for the recognition and discrimination of Cu 2+ , Hg 2+ , Fe 3+ and Fions.
Under the optimal conditions, the detection of linear relationships and limits of detection (LOD = 3 /slope) for probe L with Hg 2+ , Fe 3+ , Cu 2+ and Fare summarized in Table S1. This data suggests that probe L is also a sensitive fluorescent probe for the recognition and discrimination of these four ions. A binding analysis using the method of continuous variations established a 1:1 for L·Hg 2+ , 1:2 for L·2Fe 3+ and 1:1 for L·Cu 2+ (Fig. S10), respectively. Combining all of these observation, we proposed the possible binding mode for L·Hg 2+ , L·2Fe 3+ , L·Cu 2+ and L·Fas shown in Figure 5.
The capability of probe L to detect Hg 2+ , Fe 3+ and Cu 2+ within living cells was investigated by fluorescence imaging on a fluorescent inverted microscope. Bright-field measurements confirmed that the cells, after being treated with Hg 2+ , Fe 3+ , Cu 2+ and L, were viable throughout the imaging experiments. In the control experiment, human prostatic cancer cells (PC3) with a 10 μM probe L over 50 min led to negligible intracellular fluorescence ( Fig. 6a and  6b). When the cells were first incubated with 10 μM probe L for 50 min, and then further treated with 50 μM of metal ions (Hg 2+ , Fe 3+ or Cu 2+ ) for another 40 ~ 50 min, a significant increase in the fluorescence from the intracellular area was observed (Fig. 6c, ~ 6f). These results demonstrate that the probe is permeable to cells, binds to intracellular Hg 2+ , Fe 3+ and Cu 2+ , and emits strong fluorescent light, and thus it is highly suitable for determining intracellular Hg 2+ , Fe 3+ and Cu 2+ ions. The responses to Hg 2+ , Fe 3+ and Cu 2+ ions exhibited distinctly different colours (red and green, respectively) for the cell samples and this raises the prospect that the Hg 2+ , Fe 3+ or Cu 2+ ions could be simultaneously determined.
In conclusion, a multi-analyse probe L for the recognition and discrimination of the four ions Hg 2+ , Fe 3+ , Cu 2+ and F -, without any interference from other potentially competing ions according to their response time, max wavelength and fluorescent intensity, has been successfully obtained. Cell imaging demonstrated that probe L can be further used for monitoring intracellular Hg 2+ , Fe 3+ and Cu 2+ levels in living cells. To the best of our knowledge, this is the first case of utilizing the three dimensional scales (time, wavelength and intensity) to recognize and discriminate different ions with a single fluorescent probe; previous reports normally involve the two dimensional scales (wavelength and intensity). Our study illustrates an effective and novel strategy to differentiate multi-analyses. This work provides a promising new strategy for the simultaneous detection of ions by one simple probe.