A Fluorescent and Colorimetric Chemosensor for Hg2+ Based on Rhodamine 6G With a Two-Step Reaction Mechanism

A fluorescent and colorimetric chemosensor L based on rhodamine 6G was designed, synthesized, and characterized. Based on a two-step reaction, the chemosensor L effectively recognized Hg2+. The interaction between the chemosensor and Hg2+ was confirmed by ultraviolet–visible spectrophotometry, fluorescence spectroscopy, electrospray ionization–mass spectrometry, Fourier-transform infrared spectroscopy, and frontier molecular orbital calculations. The chemosensor L was also incorporated into test strips and silica gel plates, which demonstrated good selectivity and high sensitivity for Hg2+.


INTRODUCTION
Hg 2+ is known to be very dangerous to human health because of its extreme toxicity (Chen et al., 2012). Even extremely low levels of Hg 2+ accumulation in the human body can lead to numerous diseases in human organs (Pan et al., 2018;Peng et al., 2018;Xu et al., 2018). Thus, it is important to develop methods to detect Hg 2+ with good sensitivity and high selectivity.
Many reported Hg 2+ chemosensors were designed in one of two ways based on one-step reactions. In the first type of sensor, the interactions between a ligand and Hg 2+ results in changes in fluorescence ( Figure 1A). The main disadvantage of this type of sensor is its vulnerability to interference from other metal ions, especially Cu 2+ , Cd 2+ , and Fe 3+ Kau et al., 2012;Rao et al., 2012;Zhang et al., 2013;Kim et al., 2014;Fang et al., 2016;Alibert et al., 2017;Huang et al., 2019). In the second type of sensor, special chemical reactions (e.g., desulfurization between the ligands and Hg 2+ ) generate a new product with a different fluorescence spectrum ( Figure 1B).
In contrast, the fluorescent chemosensor L developed in this study recognizes Hg 2+ through a two-step reaction based on rhodamine 6G. The chemosensor L first undergoes a desulfurization reaction with Hg 2+ , and the resulting product then interacts with Hg 2+ (Figure 1C). As demonstrated in this study, L can selectively and sensitively detect Hg 2+ without interference from other cations, including Cu 2+ , Cd 2+ , and Fe 3+ . Moreover, test strips and silica gel plates based on L also exhibit good selectivity for Hg 2+ .

MATERIALS AND PHYSICAL METHODS
1 H nuclear magnetic resonance (NMR) and 13 C NMR spectra were recorded using a Bruker instrument at 400 MHz with tetramethylsilane as the internal standard and deuterated FIGURE 1 | Strategies for the chemosensing of Hg 2+ . Type A: The coordination between ligand and Hg 2+ . Type B: The desulfurization between ligand and Hg 2+ . Type C: The two-step reaction mechanism. dimethyl sulfoxide (DMSO-d 6 ) as the solvent. Ultravioletvisible (UV-vis) absorption spectra were recorded on a Shimadzu UV-1601 spectrophotometer. Luminescence spectra were recorded on a Horiba Fluoromax-4-NIR spectrometer. Melting points were measured on an X-4 digital melting point apparatus (uncorrected). Infrared (IR) spectra were obtained on a Nicolet 5700 FT-IR spectrophotometer. Fluorescent lifetime and fluorescence quantum yield were measured using a Horiba Fluoromax-4 spectrometer. For fluorescent lifetime measurements, the fluorescence signals were collimated and focused onto the entrance slit of a monochromator, and the output plane was equipped with a photomultiplier tube. The fluorescent decay was then analyzed by least-squares regression,  and the quality of the exponential fit was evaluated by the goodness of fit (χ 2 ). Mass spectra were recorded with a Shimadzu LCMS-IT/TOF mass spectrometer. All reagents used were of analytical grade.

RESULTS AND DISCUSSION
The spectral properties of L were studied in HEPES buffer (Figures 3, 4). When the concentration of Hg 2+ increased to 10 equivalents, a new absorption peak at 526 nm appeared (Figure 3). In the fluorescence spectrum, an emission peak appeared at 550 nm upon excitation at 526 nm (Figure 4). The Frontiers in Chemistry | www.frontiersin.org other cations did not result in the same spectral changes as Hg 2+ . Meanwhile, the color of the solution also changed significantly when 10 equivalents of Hg 2+ was added to L. This color change could be observed directly by the naked eye.
Based on competition experiments, the detection of Hg 2+ by L was not affected by the presence of other cations, including Cu 2+ , Cd 2+ , and Fe 3+ (Figures S4, S5, ESI). Surprisingly, the spectral properties depended on the ratio of [Hg 2+ ] to [L]. When the ratio did not exceed 1, the spectra did not change markedly before or after Hg 2+ was added into the solution of L. The emission peak at 550 nm did not appear (Figures S6, S7, ESI), while no change was observed in the UV-vis spectrum. The fluorescence intensity at 550 nm increased strongly when the ratio exceeded 1, and a new absorption peak at 526 nm appeared. These results are unusual in comparison to previously reported chemosensors for Hg 2+ (Figures 3, 4).
To clarify the unusual results, electrospray ionization-mass spectrometry (ESI-MS) was conducted with different ratios of [Hg 2+ ] to [L]. When the ratio did not exceed 1, the ion peak was detected at 611.3018 (m/z), corresponding to the desulfurization reaction product LO (Figure S8, ESI). This implies that L was transformed to LO under the action of Hg 2+ provided that the ratio of [Hg 2+ ] to [L] did not exceed 1 (Figure 5). Under this condition, Hg 2+ did not lead to the spirolactam ring opening of L. Thus, the fluorescent emission peak at 550 nm did not appear, nor did the absorption peak at 526 nm. Meanwhile, no color change of the solution was observed by the naked eye. The first desulfurization step was completed when [Hg 2+ ]:[L] = 1. When the ratio exceeded 1, the ion peak at m/z 813.2609 was classified as [LO-Hg 2+ +H + ] + (Figure S9, ESI). Under these conditions, Hg 2+ caused spirolactam ring opening, generating the emission peak at 550 nm and the absorption peak at 526 nm. In addition, the solution color changed from colorless to pink. After the completion of the first step, Hg 2+ transformed the closed spirolactam structure into a ring-opened amide in the second step.
The IR spectra support the above two-step reaction mechanism (Figure S10, ESI). The peak at 1,153 cm −1 , which was assigned to the stretching vibration of C=S, disappeared during the first step. The peak at 1,518 cm −1 , corresponding to the stretching vibration of C=O, vanished in the second step. Therefore, L detected Hg 2+ through a two-step reaction (Figure 5).
To understand the interaction between the chemosensor L and Hg 2+ , a titration experiment was carried out. The fluorescent  Frontiers in Chemistry | www.frontiersin.org intensity at 550 nm hardly changed when [Hg 2+ ]:[L] changed from 0 to 1, and the absorption spectrum of L did not change significantly. However, the fluorescent intensity increased sharply when [Hg 2+ ]:[L] changed from 1 to 2, and the intensity of the new absorption peak at 526 nm increased greatly. As shown in Figure 3, two inflection points were observed in the absorption spectrum of L + Hg 2+ , indicating that L might interact with Hg 2+ in two steps. The stoichiometric ratio between L and Hg 2+ was 1:1 or 1:2. The above results are in accordance with the Job plot (Figure 6 and Figures S11-S13, ESI).
The calculated frontier molecular orbitals and energy levels of L, LO, and LO-Hg 2+ confirmed the interaction between L and Hg 2+ . As shown in Figure 7, the molecular orbital structure of L was clearly different from those of LO and LO-Hg 2+ . The energy gaps between the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) in L, LO, and LO-Hg 2+ were calculated to be 0.16, 0.15, and 0.69 eV, respectively. The results indicate that Hg 2+ and L were combined via a two-step reaction, which effectively decreased the energy gap and stabilized the system.
The detection limit of L toward Hg 2+ was determined to be 0.012 × 10 −7 M (Figure S14, ESI). Moreover, the fluorescence lifetime and fluorescence quantum yield of L-Hg 2+ were measured to study the fluorescent properties of L (Table S1, ESI).
To evaluate the practical application potential of the chemosensor L, test strips and silica gel plates were prepared using L. When Hg 2+ was added to the test strips, a clear change in color from colorless to pink was observed, and the fluorescence was enhanced (Figure 8). In addition, when "Hg 2+ " was written on the silica gel plate using Hg 2+ solution, the silica gel plate underwent significant changes. The results indicate that L can be used to create smart materials for the detection of Hg 2+ in aqueous solution.

CONCLUSIONS
In summary, a chemosensor L based on rhodamine 6G was designed, synthesized, and characterized. L was demonstrated to sensitively and selectively recognize Hg 2+ via a two-step reaction, different from the one-step mechanisms by which reported chemosensors interact with Hg 2+ . The interaction between L and Hg 2+ was confirmed by UV-vis spectrophotometry, fluorescence spectroscopy, ESI-MS, IR spectroscopy, and frontier molecular orbital calculations. In addition, L could be incorporated into test strips and silica gel plates to effectively detect Hg 2+ .

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.