A new rhodamine fluorescent OFF-ON-OFF probe for selective sensing of Fe3+ and AcO- in aqueous media and living cells

A new fluorescent chemosensor for Fe3+ was developed based on a rhodamine platform. L displayed highly selective and sensitive “OFF-ON” fluorescence response and naked-eye color change to Fe3+ in aqueous solution. The resulting L-Fe3+ complex was found to act as a selective“ ON-OFF” fluorescence probe for AcOagainst common anions and cations with a Fe3+ displacement approach. The detection limits of L to Fe3+ and LFe3+ complex to AcOwere estimated to be 6.04×10-8 mol/L and 7.51×10-8 mol/L, respectively. The good biocompatibility of L enabled the investigation of fluorescent response for Fe3+ and AcOin living Ana-1 cells by confocal microscope.


1.Introduction
As an essential element for life, iron plays an important role in a wide range of biochemical processes [1], several biological functions depend directly or indirectly on the proper concentration and oxidation states of iron to maintain the homoeostatic mechanism of biosystem [2,3]. Its deficiency and excess can induce a variety of diseases [4,5]. Therefore, it is highly desirable to develop fluorescent probes for the real-time monitoring of iron ions in environmental and living systems [6,7] . Due to great sensitivity, fluorescent chemosensors combining ionophores and fluorophores are commonly considered superior [8][9][10] . In recent years, rhodamine has been attracted a great deal of attention due to its excellent properties, including good photostability, long emission wavelength, high fluorescence quantum and so on [11][12][13] . However, for the majority of rhodamine fluorescent probes, their anti-interference ability is poor, which are usually chelate with two or more metal ions simultaneously [14] . Due to the interference of Cr 3+ , Al 3+ , Cu 2+ in the identification process of Fe 3+ [15][16][17][18] , rhodamine fluorescent probe exhibited low selectivity for Fe 3+ [19,20] .
The recognition and sensing of anions by proper design of anion receptors is currently of major interest because anions play a fundamental role in chemical and biological processes. Various kind of anions such as F -, Cl -, I -, PO4 3-, CH3COO -, etc. play a major role both in environmental and biological systems [21][22][23] . Among the above anions, AcOreceived the most attention from chemists because of its unique properties, the detection of AcOwith the help of easily synthesized receptor and minimal instrumental assistance is desirable towards practical applications [24,25] . Recently, rhodamine-cation complex solution has been documented for the recognition of various anions [26][27][28] . This intrigued us to design a novel Fe 3+ -fluorophore complex to develop a AcO --specific fluorescence chemosensor.
In this work, the allyl group was introduced into p-Hydroxybenzaldehyde to improve the charge distribution of probe, herein we designed and synthesized a new turnon fluorescence sensor based on rhodamine for the quantification of Fe 3+ . The in situ formed L-Fe 3+ complex solutions exhibited a sensitive response to AcOvia a turn-off response by the displacement strategy. The reversible "off-on-off" fluorescence sensor in solution showed the good application in live Ana-1 cell imaging.

Results and discussion
The synthetic route for compound L was shown in Scheme 1. The structure of L was confirmed by NMR, mass spectra ( Fig.S1-S5). The detailed experimental procedures and the characterization of the compound L were described in the experimental section.
The recognition profiles of compound L were investigated in CH3CN/H2O solutions by UV-vis spectroscope toward various heavy metal and transition metal cations, including Ca 2+ , Cd 2+ , Cu 2+ , Hg 2+ , Mn 2+ , Ni 2+ , Pb 2+ , Zn 2+ , Fe 3+ , Na + , Al 3+ , Mg 2+ , Ag + , Cr 3+ . As presented in Fig. 1, free probe L showed an absorption band at 558nm, when 10 equiv. of Fe 3+ was added to the solutions of probe L, the absorption intensity was significantly enhanced, the change in spectroscope was come along obvious color change from colorless to pink, Differently, nearly no significant adsorption changes could be observed in the presence of other interested metal ions. These results clearly suggested that L can serve as a "naked-eye" Fe 3+ indicator in aqueous media. In addition, we have tested the fluorescence properties of L toward a series of metal ions such as Cu 2+ , Pb 2+ , Fe 3+ , Fe 2+ , Hg 2+ , Al 3+ , Cr 3+ , Ni 2+ , Mn 2+ , Co 2+ , Zn 2+ , Ag + , Ba 2+ , Cd 2+ and Na + (100μM) in CH3CN/H2O buffer (3:7, v/v, 20mM, pH=7.2). As shown in Fig. 2, fluorescence probe L alone and other metal ions all displayed weak fluorescence emission bands except for Fe 3+ , the addition of Fe 3+ induced a remarkable fluorescence enhancement (59-fold), whereas the addition of other metal ions to the solution L exhibited no obvious change in the fluorescence spectra. Meanwhile, the emission color of solutions can be clearly observed to change from colorless to red-orange under illumination with 365 nm light in presence of Fe 3+ , indicating that there were some interaction between L and Fe 3+ . The specific response of L towards Fe 3+ should be attributed to be based on the opening function of the spirolactam ring [29][30][31] . Compound L can be considered as a new off-on fluorescent chemosensor for Fe 3+ .
The interference of the potentially competing metal ions toward Fe 3+ detection was also investigated. As shown in Fig. 3, chemosensor L was treated with 10 equiv. of Fe 3+ and 10 equiv. of other coexistent metal ions such as Cu 2+ , Pb 2+ , Fe 2+ , Hg 2+ , Al 3+ , Cr 3+ , Ni 2+ , Mn 2+ , Co 2+ , Zn 2+ , Ag + , Ba 2+ , Cd 2+ and Na + . As a result, no interference was observed for the detection of Fe 3+ by L, these results manifest the recognition of Fe 3+ was not interfered by these competing metal ions.
Fluorescence titration of L (10μM) with Fe 3+ was performed in CH3CN/H2O (v/v=3:7) solution. As shown in Fig.5, the fluorescence intensity increased gradually in the presence of increasing amounts of Fe 3+ (0-10equiv.). Job plot was utilized to determine the binding stoichiometry of the L-Fe 3+ complex, it revealed that L and Fe 3+ formed a complex in 1:1 stoichiometry (Fig.4) [32] . Hildebrand-Bebesiplots gave binding constants of 2.9×10 4 M -1 in HEPES buffer [33] (Fig. 5). The detection limit for Fe 3+ was estimated to be 6.04×10 -8 M based on a 3σ/slope analysis (Fig. S6) [34] .Which was sufficiently low to detect submicromolar concentration of the Fe 3+ , implying that L had great potential for use in the development of Fe 3+ sensor materials.   Furthermore, the probe L was found to have a short respone time towards Fe 3+ . As shown in Fig. S7, the fluorescence intensity of L rapidly enhanced after Fe 3+ (100μM) was added, reached its maximum value in 1 min and stabilized thereafter, indicating that the L could be used for the real-time detection of Fe 3+ .
The recognition of L-Fe 3+ complex towards anions and nucleotides were then evaluated. As shown in Fig. 6, the fluorescence intensity of the L-Fe 3+ at 580nm was gradually decreased with the addition of increasing concentration of AcO -(0-2equiv.), and the color of receptor L-Fe 3+ solution showed a dramatic color change from pink to colorless, indicating that the decomplexation of Fe 3+ by AcO -. However, other anions such as S 2-, F -, Cl -, Br -, NO3 -, HSO4 -, H2PO4 -, PO4 3-, SCN -, HPO4 2and Cys were found to hardly induce any variation either in visible color change or in the absorption spectra (Fig. 7). In addition, fluorometric titration assays detected the reversible detection of AcO -. As shown in Fig.8, "OFF-ON-OFF" fluorescence changes were observed by the alternative additions of Fe 3+ and AcOto the solution of L and L-Fe 3+ respectively, indicating that L can be developed as a reversible fluorescence chemosensor for Fe 3+ and AcO -. The detection limit of L-Fe 3+ for AcOwas determined to be 7.51× 10 -8 mol/L (Fig. S8).  The effect of pH on the fluorescence of probe L to Fe 3+ and L-Fe 3+ complex to AcOwas evaluated. As shown in Fig. 9, L and L-Fe 3+ were all weakly fluorescent over a wide pH range (pH=4.0-10.0). Meanwhile, probe L displayed an efficient fluorescence response to Fe 3+ in the pH range of 5.0-8.0. It can be seen that the fluorescence intensity of L-Fe 3+ is evidently enhanced (pH≥5) and reaches its maximum at pH 7. The results indicated that the L can be used as a chemosensor for Fe 3+ detection and the L-Fe 3+ ensemble could work for the detection of AcOin physiological conditions.
The proposed displacement strategy for the AcOdetection was further supported by the results of mass spectra studies, FT-MS spectra of L showed a molecularion peak [L+H + ] + at m/z 601.31426 (Fig. S3). When the Fe 3+ ion was added into the solution of L, the peak at m/z 718.4273 is assignable to [L _ 2H + +Fe 3+ +NO3 -+H + ] + species (Fig. S4). This result confirmed that 1:1 stoichiometry complex between L and Fe 3+ . In the presence of AcO -, FT-MS spectra displayed a molecularion peak at m/z 601.31695 again (Fig. S5), indicating that the decomplexation of Fe 3+ ion in the presence of AcOled to the liberation of fluorophore, L. Scheme 2. The proposed mechanism for Fe 3+ and AcO − detection by L The cytotoxicity of L toward the Ana-1 cells was investigated by the reduction activity of the methyl thiazolyl tetrazolium (MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) assay. As shown in Fig. S9, the cellular viability was estimated to be approximately 89.5% after incubation with L (20μM) for 24 h, which demonstrates that L has low cytotoxicity and biocompatibility to living cells. Due to the good spectroscopic properties and biocompatibility of the probe, L was applied for fluorescence imaging in living Ana-1 cells. As shown in Fig. 10, Ana-1 cells incubated with 5μM L for 30 min at 37℃ gave no intracellular background fluorescence [35] (Fig. 10a). Moreover, no intracellular fluorescence was detected for Ana-1 cells with 50μM FeCl3 in the growth medium for 3 h at 37℃. However, as shown in Fig. 10b, bright intracellular fluorescence was observed for Ana-1 cells with 50μM FeCl3 in the growth for 3 h at 37℃ and then staining with L under the same loading conditions. After treating the cells with 25μM NaOAc for another 3 h at 37℃, the intense red fluorescence of the cells disappeared (Fig.  10c). It can be seen from the bright field transmission measurement after Fe 3+ and L incubation (Fig. 10d) that the Ana-1 cells were viable throughout the imaging experiments. These facts implied that L was membranepermeable and could be used for detecting Fe 3+ in biological samples.

Materials and instrumentation
All reactants and chemicals were obtained from Aladdin and used without further purification. Thin layer chromatography (TLC) was carried out on alumina backed plates coated with Merck gel GF254. Mass spectral data were obtained on a Solaril X70 FT-MS. 1 H NMR and 13 C NMR were recorded at room temperature on a Bruker Avance-500 NMR spectrometer. Chloroform (CDCl3) was used as solvent and tetramethylsilane (TMS) as internal standard. Fluorescence spectra measurements were recorded with a Hitachi F-4500 spectrofluorometer. A Techcomp UV-8500 spectrophotometer was used for Absorption measurements.

Synthesis and Characterization
The substituted benzaldehyde (3) was prepared according to the literature method [36] . The unique ligand L was prepared via the reaction of rhodamine hydrazine and 3 in methanol. (

Conclusion
In summary, a new fluorescent probe L was designed and synthesized. L exhibited extreme selectivity for Fe 3+ over other metal ions in aqueous environment. Furthermore, the in situ generated nonfluorescent L-Fe 3+ complex was employed for the specific recognition of AcOby fluorescence turn-off signaling through a displacement process. Confocal microscopy imaging implied that L can be potentially used as a promising candidate for Fe 3+ and AcOimaging in Ana-1 cell.