Recognition Preference of Rhodamine Derivative Bearing Phthalimido Gly for Hg 2 + by UV-Vis and Fluorescence Spectroscopy

An effective Hg-speci�c probe 1 was designed and prepared based on Phthalimido Gly modi�ed rhodamine B. e sensing behavior of probe 1was studied by �V-Vis and �uorescence spectroscopy. Probe 1 showed excellent high selectivity and sensitivity towardsHg over commonly coexistentmetal ions in neutral solution,which could be attributed to theHg-triggered ring opening of the spirolactam of the rhodamine moiety and the formation of a 1 : 1 1-Hg complex. e limit of detection (LOD) based on 3δδblank/kk was calculated to be 2.8 × 10 M, as well as an excellent linear relationship with the concentration of Hg in the range from 0.1 × 10 to 1.0 × 10mol/L (RR = 0.98927). In addition, the effects of pH, coexisting metal ions, and the reversibility were investigated in detail.


Introduction
e design and development of �uorescent chemosensors for detection of environmentally and biologically important metal cations (such as Hg 2+ , Fe 3+ , and Al 3+ ) are currently of signi�cant importance [1][2][3][4][5].More speci�cally, sensors directed toward the detection and measurement trace amounts of mercury ions which have enjoyed particular attention.e Hg 2+ ion is considered highly dangerous because both elemental and ionic mercury can be converted into methyl mercuries by bacteria in the environment, which subsequently bioaccumulates through the food chain.Moreover, the extreme toxicity of mercury and its derivatives result from its high affinity for thiol groups in proteins and enzymes, leading to the dysfunction of cells and consequently causing health problems [6][7][8].Current techniques for Hg 2+ screening, �uorescence chemosensors offer a nondestructive and prompt detection of Hg 2+ by a simple �uorescence enhancement (turn-on) or quenching (turn-off) response in biological, toxicological, and environmental monitoring, due to its simplicity and distinct advantages in sensitivity and selectivity to recognition of Hg 2+ [9][10][11][12][13][14].
Among numerous chemosensors, Rhodamine B and its derivatives are a kind of excellent candidate for the construction of an off/on-type �uorescent chemosensor and well known for their desirable properties, including excellent spectroscopic properties, high extinction coefficient (>75,000 cm −1 M −1 ), high �uorescence quantum yield, long absorption and emission wavelength elongated to the visible region [15][16][17][18].e mechanism is based on the switch "offon" of the spirocyclic moiety mediated by guest.On binding guest to the receptor, the spirocyclic form of rhodamine B which is colorless and non�uorescent, whereas ring opening of the corresponding spirolactam induced by metal ions gives rise to strong �uorescence emission and a pink color [19][20][21][22].
Consequently, the design and development of �uorescent chemosensors for monitoring the level of Hg 2+ in environmental and biological samples have attracted a great deal of attention.Herein, we introduced a rhodamine B derivative (probe 1) bearing Phthalimido Gly group.In our condition, probe 1 exhibited prominent absorption and �uorescence enhancements to Hg 2+ ions with a particular selectivity and excellent sensitivity and could be used for naked-eye detection.

Experimental Section
2.1.Materials and Instrumentation.All solvents and reagents were purchased from commercial sources and used as received without further puri�cation.Solutions of metal ions were prepared with metal nitrate salts.Doubly-distilled water was used for all experiments.UV-Vis spectra were obtained on a Purkinje General TU-1901 UV/Vis spectrometer.e �uorescence spectra were recorded on a Hitachi F-4500 spectro�uorimeter with a quartz cuvette (path length, 1 cm).IR data were taken in KBr disks on TENSOR37 Fourier-transform infrared spectrometer.NMR spectra were recorded on a Bruker 300 MHz spectrometer with TMS as an internal standard and CDCl 3 as solvent.e stock solution of 1 (2.0 mmol/L) was prepared by dissolving the requisite amount of it in methanol.Standard test solutions were prepared by appropriate dilution of the stock solution.A solution of the nitrate salts of the respective ions Na + , K + , Mg 2+ , Mn 2+ , Fe 3+ , Ca 2+ , Zn 2+ , Ag + , Ba 2+ , Pb 2+ , Cu 2+ , Cd 2+ , Ni 2+ was used to evaluate the metal ion binding properties of 1 (1.0 × 10 −5 M) in C 2 H 5 OH-water (4 : 6, v/v) buffered with HEPES, pH = 7.0.e solution was allowed to stand for 10 min at room temperature (25 ∘ C) before the absorption/�uorescence measurement was made.For �uorescence measurements, excitation was provided at 556 nm, and emission was collected from 450 to 700 nm.e excitation and emission slit widths were 5 nm and 10 nm.

Determination of Binding
Constants.e binding constant was calculated from the absorption intensity titration curves according to Benesi-Hildebrand equation as follows [23]: where  and  0 represent the absorbance of 1 in the presence and absence of Hg 2+ , respectively,  max is the saturated absorbance of 1 in the presence of excess amount of Hg 2+ ; [c] is the concentration of Hg 2+ ion added (mol/L).

Synthesis of Probe 1. (See Scheme 1)
. RhE was synthesized from rhodamine B by reaction with ethylenediamine in ethanol following a published procedure in a yield of 70% [24]. 1 was prepared by one-step of rhodamine ethylenediamine with Phthalimido Gly chloride at 0-5 ∘ C. Brie�y, rhodamine ethylenediamine (1.88 g, 4 mmol) was dissolved in 20 mL dichloromethane in a 100 mL �ask.An excess of triethylamine was added and 15 mL dichloromethane solution of Phthalimido Gly chloride (0.89 g, 4 mmol) was added to this solution.e reaction mixture was stirred 2 h

Results and Discussion
3.1.UV-Vis Spectral Characteristics.Like most of the spirocycle rhodamine B derivatives, due to its spirolactam form, the absorption spectra of free 1 (10 × 10 −5 M) remained colorless and exhibited very weak absorbance from 450 to 650 nm and found to be very stable in C 2 H 5 OH-water (4 : 6, v/v) buffered with HEPES pH = 7.0 solution system for more than one week.Upon incremental addition of Hg 2+ (0−20×10 −5 M) to probe 1 solution (10 × 10 −5 M), the peak around 561 nm was formed and its intensity increased gradually with increasing Hg 2+ concentrations (Figure 1), suggesting the formation of the ring-opened form of 1.Moreover, the titration solution exhibited an obvious and visual color change from colorless to pink, which could be easily detected by the "naked-eye" (Figure 1, inset (a)).
For further determination the stoichiometry between Hg 2+ and 1, Job's plot analyses were also used.According to the continuous variations with a total concentration of [Hg 2+ ] + [1] as 1.0 × 10 −5 M. (Figure 1, inset (b)).When the molar fraction of Hg 2+ was 0.5, the absorbance exhibited a maximum, which demonstrates the 1 : 1 complex between 1 and Hg 2+ .

Fluorescence Spectral
Characteristics.e sensing behavior was investigated by the �uorescence measurement in C 2 H 5 OH-water (4 : 6, v/v) buffered with HEPES pH = 7.0 solution upon excitation at 556 nm.As shown in Figure 3(a), upon the gradual addition of Hg 2+ (0-2 × 10 −5 mol/L), an emission band peaked at 581 nm signi�cantly increased in �uorescence intensity.e titration reaction curve showed a steady and smooth increase until a plateau was reached (15.0 M Hg 2+ ), e chemosensor exhibited very efficient �uorescence responding, which is attributed to the formation of 1-Hg 2+ complex as a result of which the non�uorescent spirocyclic form of the receptor get opened leading to the formation of strongly �uorescent ring-opened amide form.
In addition, to see its practical applicability, the detection limit of 1 for Hg 2+ was evaluated.e �uorescence intensity of 1 (1.0 × 10 −5 mol/L) at 581 nm was found to increase linear relationship with the concentration of Hg 2+ in the range of 01 × 10 −6 to 10 × 10 −6 mol/L ( 2  0.98927) (Figure 3. inset(a)).e detection limit is then calculated to be 2.8 × 10 −8 M based on 3 blank / [25], where  blank is the standard deviation of the blank measurements,  is the slope between �uorescence intensity versus sample concentration.e results indicated that probe 1 is sensitive enough to detection of trace mercury ion in the environment.

Effect of pH Value.
To study the practical applicability, the sensitivity of the Hg 2+ �uorescent probe 1 toward variations in the sample pH was examined in the absence and presence of the Hg 2+ ion. Figure 4 clearly indicates that the free 1, which has weak �uorescence between pH 7.0 and 1�.0.However, the �uorescence intensity become strong when pH value below 7.0.It is due to that the ring opening of rhodamine took place for the strong protonation.Under identical condition, while addition of the Hg 2+ ion, 1 showed a meaningful response between pH 5.0 and 9.0, because it can lead to a remarkable increase of �uorescence.Finally, in order to eliminate the in�uence of pH, the HEPES buffer system, at a physiological pH value 7.0, was chosen for the �uorescence intensity determination.

Selectivity and Completion.
Selectivity is an important parameter to evaluate the performance of a chemosensor.As shown in Figure 5, the absorbance and �uorescence spectra changes of Rh-G caused by Hg 2+ and various competitive  Moreover, in order to verify the high selectivity of 1 toward Hg 2+ , competition experiments in the presence of miscellaneous competitive metal ions (above mentioned) were conducted.e enhancement in �uorescence intensities resulting from the addition of the Hg 2+ was hardly in�uenced by the subsequent addition of excess metal ions (Figure 6).It is likely that the addition of Hg 2+ induces the carbonyl oxygen atom of spirolactam to coordinate with Hg 2+ resulting in the opening of spirolactam ring that leads to the development of pink colour and enhancement in �uorescence intensity.

e Reaction Mechanism and
Reversible. e chemosensor is the most likely to chelate with Hg 2+ via its three oxygen on the three carbonyl groups (Scheme 2).To test whether the proposed complex could be reversed, we added Na 2 S to the solutions of 1-Hg 2+ species.Upon addition of Na 2 S, �uorescent emission intensity of the system was quenched (Figure 7), and the color of the mixture of 1 and Hg 2+ changed from pink to almost colorless, indicating that Na 2 S replaced the receptor 1 to coordinate Hg 2+ .us, these �ndings indicated that probe 1 can be classi�ed as a reversible chemosensor for Hg 2+ .

Conclusions
In summary, we have synthesized a new rhodamine-based probe 1 linked with Phthalimido Gly for the detection of Hg 2+ ions, which displayed Hg 2+ via a 1 : 1 binding mode enabled the spirolactam ring (non�uorescence) to the ringopened amide (�uorescence), a desirable "off-on" mode.Moreover, its �uorescence intensity was enhanced in a linear fashion with a Hg 2+ concentration from 0.1 × 10 −6 to 1.0 × 10 −6 mol/L, with a detection limit of 2.8 × 10 −8 M. us, probe 1 also enables the "naked-eye" detection of Hg 2+ over other commonly coexistent metal ions (even those that exist in high concentration).

S 1 :
Synthetic routes of the probe 1.

S 2 :F 7 :
Proposed possible binding mode of probe 1 with Hg 2+ .e emission spectra of 1 (10.0 M C 2 H 5 OH-water solution 4 : 6, v/v pH = 7.0) in the presence of 1 equiv. of Hg 2+ and 5 equiv. of Na 2 S.
2+only.In contrast, other metal ions developed no signi�cant absorption and �uorescence intensity changes under the identical conditions.ese results indicate that probe 1 has a remarkable selectivity towards Hg 2+ and could serve as a "naked-eye" chemosensor selective for Hg 2+ in the aforementioned buffer solution.