The development of sensors based on the ion-induced changes in fluorescence is particularly attractive . Due to the unique properties, rhodamine is a promising scaffold for the design of reversible and selective probes . Heavy and transition metal ions are toxic ions known as lacking any vital or beneficial effects in biological system and damaging the ecological environment , among which Hg2+ is an extremely toxic rare element in the earth's crust and its toxicity has made public , considerable attention has been paid to the development of new fluorescent chemosensors [5, 6] for the detection of mercury and mercuric salts with sufficient selectivity [7, 8]. At present, detection of Hg2+ mainly uses spectral method, including atomic (absorption, emission, and fluorescence) spectrum, spectrophotometric and electrochemical method [9, 10], etc. Compared with traditional detection methods, fluorescence approach based on molecular chemosensors is particularly significant owing to its high sensitivity, selectivity, low cost, non-destructive analysis, operational simplicity, and their potential applications in analytical chemistry and biomedical sciences [11, 12]. Hg2+ can couple with spin orbit of fluorescent molecules leading to fluorescence quenching . Therefore, a lot of mercury fluorescent probe is based on fluorescence quenching mechanism, and this sensitivity of the turn-off probe is below the turn-on probe. Besides, some rhodamine B derivatives have also been used as fluorescent chemosensors, but the structure changes of water-soluble "off-on" fluorescent probe were confirmed merely by NMR, IR, and ESI mass data [14, 15] without calculation.
Herein, according to rhodamine-based probes for mercury ion detection reported [16, 17], two different rhodamine derivatives were synthesized (RA1 and RA2) towards Hg2+ based on the equilibrium between spirolactam (nonfluorescent) and ring-open amide forms (highly fluorescent) induced by specific chemical species at room temperature [18-21]. They are used as a fluorescent signal transducer due to their tremendous photophysical properties such as extended absorption and emission wavelengths, high fluorescence quantum yield and large molar absorption coefficient [22-25]. The signal mechanism follows a straightforward protocol, after binding of the analyte (metal ion) to spirolactum ring in which the rhodamine core breaks with an excellent enhancement in the fluorescence intensity and develops a strong colour and naked eye detection [26, 27]. Chemosensor RA2 displayed a high selective and sensitive colorimetric change and fluorescence enhancement response to Hg2+.Ⅱ. EXPERIMENTS
The structure of intermediate RBH, title compounds RA1 and RA2 were characterized by 1H NMR (Fig.S1, S2, and S3), 13C NMR (Fig. S2 and S3) and EI-MS (Fig.S1, S2, and S3) in supplementary materials. The results were in good agreement with the structure shown in Scheme S1, S2, and S3 (in supplementary materials). Fluorescence and UV-Vis studies were performed using a 10 μmol/L solution of RA1 and RA2 in a CH3CN/H2O (1:1, V/V) solution with appropriate amounts of metal ions.Ⅲ. RESULTS AND DISCUSSION A. Steady-state optical properties
Compared with ion mixture RA1-Hg2+, RA2-Hg2+ had higher fluorescence quantum yield (Table S1 in supplementary materials). RA2 exhibited twenty times enhancement of fluorescence intensity at peak wavelength λmax=580 nm in the presence of 10 equiv. Hg2+ as shown in Fig. 1. It was discovered that hydroxy benzene of rich electron was beneficial to the chelate of Hg2+ with sensors through various tests. Owing to more stable structure, RA2 showed better sensing property, so we selected RA2 as a representation example to expatiate in the following discussion.
The effect of the reaction media for the binding of RA with Hg2+ was studied, and the results are shown in Fig.S4 (in supplementary materials). It was found that solvent has great effect on the coordination reaction. In pure water, the interaction between Hg2+ and probe is not obvious, fluorescence intensity of the system is very weak compared with other solvent system add acetonitrile. When the volume ratio between 1:9 and 9:1, probe can identify Hg2+ well. When the coordination reaction was performed in acetonitrile-water solution of 1:1 (V/V), the highest fluorescence intensity F values were obtained, indicating that volume ratio of acetonitrile to water=1:1 is favorable for fluorescent measurement . With the increase of acetonitrile, fluorescence intensity hardly changes. However, the increasing dosage of organic reagents are adverse to biological environment. Therefore, volume ratio of acetonitrile-water was selected as 1:1 for fluorescent assay and the colorimetric assay, respectively.B. UV-Vis and stoichiometry complexation
As shown in Fig. 2, UV-Vis spectrum of compound RA2 (10 μmol/L) exhibited only very weak bands over 400 nm. Addition of 10 equiv. Hg2+ into solution immediately resulted in a significant enhancement of absorbance at about 556 nm simultaneously the color changed into purple . Under the identical condition, no obvious response could be observed upon the addition of other ions including Zn2+, Mg2+, Ca2+, Cd2+, Cu2+, Pb2+, Cr3+, Ba2+, Ni2+, Fe3+, Mn2+, K+, Li+, Ag+, Co2+, Na+, Cu2+, and Al3+, which caused a mild effect compared to Hg2+. This phenomenon demonstrated that compound RA could serve as a selective "naked-eye" chemosensor for Hg2+. (Fig. 2 and Fig.S5 in supplementary materials).
The Hg2+ binding stoichiometry of RA can be determined from titration and the Job plot . A plot of [Hg2+]/([Hg2+]+[RA1]) versus the molar fraction of Hg2+ was provided in Fig.S6 (supplementary materials). The absorbance reached a maximum when the ratio was 0.5, indicating a 1:1 stoichiometry of the Hg2+ to RA in the complex.C. Selectivity
As shown in Fig. 3, RA2 exhibited a very weak fluorescence in the absence of metal ions. When 10 equiv. Hg2+ was added to a 10 μmol/L solution of RA2 in CH3CN/H2O (1:1, V/V), a remarkably enhancement of fluorescence spectrum was observed. The fluorescence enhancement of Hg2+ to compound RA2 was as high as 20 times. Under the same condition, a mild fluorescence enhancement factor was also detected for Cu2+ and Al3+, but other ions showed no obvious changes on fluorescence intensity and color. Moreover, we also confirmed the competitive experiments that the background metal ions showed very low interference with the detection of Hg2+ water solution (Fig. 4) .D. Sensitivity
To further investigate the interaction of Hg2+ and RA, an fluorescence titration experiment was carried out (Fig. 5(a), Fig.S7(a) in supplementary materials). Add 20 μL Hg2+ into 2 mL solution in turn and measure fluorescence intensity. Seen from Fig. 5, fluorescence was weak without Hg2+, fluorescence intensity of RA2 increased significantly with the increase of Hg2+ ion concentration, the maximum emission wavelength redshift from 575 nm to 581 nm, proving that RA2 changed from spirolactam to ring-open amide (large fluorescent conjugated system). The solution changed from colorless to pink. Whether the solution contains Hg2+ could be identified with naked eyes . Generally, the detection limit of metal ions is needed for fluorescence sensor. Fluorescence enhancement efficiency is closely associated with the Stern-Volmer constant (KSV) . The equations of the enhancement effect were fitted, and the related parameters are listed in Table Ⅰ. A signal-to-noise ratio S/N=3 is generally considered to be the limit of detection  (LOD=3σ/KSV). Under optical conditions, RA2 showed larger red-shift value of 10 nm, the linear fluorescence intensity response of compound RA2 was between 20 and 100 μmol/L (Fig. 5(b)), and the LOD for Hg2+ was measured to be 0.21 and 0.14 μmol/L of RA1 and RA2 respectively, which illustrated the high sensitivity of probes. K, the association constant value of the RA2-Hg2+ complex was calculated to be 1.74×104 (mol/L)-1, which was greater than K of RA1-Hg2+ complex 3.46×103 (mol/L)-1, both two showed a fine linear relationship (Fig. 5(c), Fig.S7(c) in supplementary materials) by the Benesi-Hildebrand plot .E. Mechanism
To investigate the Hg2+ enhancement mechanism, IR spectra of RA and RA+Hg2+ were taken in KBr disks (Fig.S8, S9 in supplementary materials). The peak at 1680 cm-1, which corresponds to the C=N absorption disappeared upon the addition of Hg2+. This supported the notion that the C=N group of RA is involved in the coordination of metal ions .
The adaptability has been determined using the lifetime of RA and Hg2+ via a time resolved fluorescence spectrofluorometer. The fluorescence lifetime was measured by single photon counting at an excitation 460 nm of the NanoLED source. The decays of RA and RA+Hg2+ were found to be two exponentials and monoexponential which were on behalf of two and one luminescent mechanism respectively. The lifetime decays in the absence and presence of Hg2+, which are shown in Fig. 6 and Fig.S10 (supplementary materials). The lifetime was 1.63 and 4.18 ns (XSQ=1.27) for RA2 lonely and 1.82 ns (XSQ=1.78) with Hg2+, indicating that structure of RA2 changed after chelation of Hg2+. When molecules were in excited state, lone pair electrons of two electron donor (ethylenediamine) moved to the acceptor (central carbon atom with a positive charge). Meanwhile alkyl rotated around C-N bond leading to orthogonal state with aromatic ring plane, the original conjugated system was damaged (as red circle marked). So RA2 owned two fluorescence lifetimes. After adding Hg2+, the central carbon atom was broken and the whole molecule formed a large conjugated system, which resulted in only a fluorescent lifetime .
In addition, the Hg2+-adding experiments were conducted to examine the reversibility of this reaction and the result was shown in Fig.S11 in supplementary materials. When ethylenediaminetetraacetic acid (EDTA), 3 equiv. of Hg2+) was added to the RA+Hg2+ of CH3CN/H2O solution, the fluorescence intensity at 580 nm was decreased and further addition of 10 equiv. Hg2+ could almost recover the fluorescence.
The HR-MS of RA2+Hg2+ in CH3CN/H2O was also conducted (Fig.S3 in supplementary materials). An unique peak at m/z=559.29 corresponding to [R6+H]+ was clearly observed when 1 equiv. of Hg2+ was added to RA2, whereas RA2 without Hg2+ exhibited peaks only at m/z=561.28, which corresponded to [RA2+H]+ (Fig.S5 in supplementary materials).
Both UV-Vis and fluorescence data lead to a significant OFF-ON signal. From the molecular structure and spectral results of RA, an reversible fluorescent chemodosimeter for Hg2+ was constructed as shown in Fig. 7 and Fig.S12 in supplementary materials. Firstly, the addition of the Hg2+ ion induced a ring opening of the spirolactam of rhodamine took place. Then, RA-Hg was hydrolyzed into carboxylation. And the first reversible reaction was certified by theoretical calculation, while next hydrolysis reaction was confirmed by mass spectrometry (Fig.S2 and S3 in supplementary materials).
To interpret basic sensing mechanism, calculations based on time-dependent density functional theory (TD-DFT) were performed on this system (Gaussian 03 program). The structures of RA2 and R5 were optimized and the calculated results demonstrate that the free energies of R5 is lower than those of RA2 and Hg2+ (△G=-279 kcal/mol), indicating that this open loop step is an exothermic process.
For compound RA2, the highest occupied molecular orbital (HOMO) is mainly located on the xanthene ring and the lowest unoccupied molecular orbital (LUMO) is on the upper portion of hydrazine, in addition, the lowest energy transitions of RA2 and R5 come from HOMO to LUMO. The HOMO and LUMO energies of R5 are lower than those of RA2 and the energy difference between HOMO and LUMO of R5 (△E=0.3972 eV) is smaller than that of RA2 (△E=3.168 eV) (Fig. 9). Addition of Hg2+, the energy gap between the HOMO and LUMO is greatly decreased . In addition, DFT calculations were carried out for the geometry optimizations of the probe. From the optimized structure (Fig. 8) and the bond lengths of RA2 (C1-N1, 1.39 Å; N1-N2, 1.37 Å; N2-C3, 1.30 Å) and R5 (C1'-N1', 1.38 Å; N1'-N2', 1.33 Å; N2'-C2', 1.32 Å), we can conclude that hydroxybenzaldehyde was conjugated to the fluorophore. As a result, ring opening of the spirolactam can alter the push-pull effect of the fluorophore greatly.F. Application
Many fluorescent sensors for Hg2+ detection could only be performed in solution, which would limit their applications under special circumstances. To demonstrate the practical application of our sensor, we prepared the test strips of sensor RA2. It was easily prepared by immersing a filter paper into the CH2Cl2 solution of RA2 (1 mmol/L) and then drying in air. Next, to different Hg2+ concentration solutions (0, 1.0×10−4, 1.0×10−3, 1.0×10−2 mol/L), these strips were immersed for 5 s and taken out of the solution. As depicted in Fig. 10, the color of the test strips changed from colorless to purple and deepened gradually with the increasing of Hg2+ concentration. Thus, these strips could be conveniently handled at any moment for the detection of Hg2+ ions.
Hydrogels have been used for environmental applications: absorption and separation in many studies . Hg2+ ion selective and naked-eye colorimetric copolymeric sensor hydrogels were synthesized using N-isopropyl acrylamide (NIPAM) and acrylamide (AAm) as the primary monomer by crosslinking and curing for 10 min. Here we designed colorimetric chemosensors for visual detection of Hg2+ ion based on rhodamine hydrogel. Used as naked-eye sensors for Hg2+ ion in aqueous media, it was observed that color change of the hydrogels in 10 μmol/L Hg2+ solution began in a few seconds. As seen in Fig. 11, gel acts as a naked-eye and fluorescent light responsive sensor for Hg2+ ion under visible light and light at 365 nm. The simple and convenient test paper and hydrogel may provide an easy way to detect Hg2+ in our daily life.Ⅳ. CONCLUSION
We synthesized two fluorescent chemodosimeters RA1 and RA2 based on rhodamine schiff-base conjugate. High sensitivity and selectivity for Hg2+ recognition in CH3CN/H2O (1:1, V/V) solutions and hydroxy benzene of rich electron was beneficial to the chelate of Hg2+ with sensors were demonstrated. The colorimetric and fluorescent response to Hg2+ can be conveniently detected even by the naked eye, which provides a facile method for visual detection of Hg2+. In the absence of the cations, these probes adopt a spirocyclic form, which was colourless and nonfluorescent, whereas, after addition of metal ions, the spirocyclic ring open via a 1:1 stoichiometric coordination or reversible chemical reaction in short time. Meanwhile, the unapparent changes of fluorescence lifetime decay suggested that turn-on process of probe combined with Hg2+ appeared to be a static mechanism. Hydrogel acts as colorimetric chemosensors through naked-eye and fluorescent light response for Hg2+ ion under visible light and light at 365 nm.
Supplementary materials: Synthesis and characterization of compound RBH, RA1, RA2, and additional spectra of RA1 are shown in Fig. S1-S3, S12. Synthesis and characterization of compound RBH, RA1, RA2 (Fig.S1-S3, S8, S9), effect of solvent ratio, pH, reaction time on fluorescence intensity (Fig.S4, S6), UV absorption and fluorescence emission upon addition of different metal ions (Fig.S5, S7, S11), lifetime decays and theoretical model (Fig.S10, S12) are also shown.Ⅴ. ACKNOWLEDGMENTS
This work was supported by the Fundamental Research Funds for the Central Universities (KYLX15\_0125) and National Major Scientific Instruments and Equipment Development Projects (2014YQ060773).
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