Synthesis and evaluation of a novel rhodamine B-based ‘off-on’ fluorescent chemosensor for the selective determination of Fe3+ ions

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Synthesis and evaluation of a novel rhodamine B-based ‘off-on’ fluorescent chemosensor for the selective determination of Fe3+ ions Hailang Chen a,c , Xiaofeng Bao a,∗ , Hai Shu a , Baojing Zhou c , Renlong Ye c , Jing Zhu b,∗ a b c

School of Environmental and Biological Engineering, Nanjing University of Science and Technology, 200 Xiaolinwei, Nanjing 210094, PR China Department of Pharmacy, Nanjing University of Chinese Medicine, 138 Xianlin Dadao, 210023, PR China School of Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210094, PR China

a r t i c l e

i n f o

Article history: Received 4 June 2016 Received in revised form 24 September 2016 Accepted 27 September 2016 Available online xxx Keywords: Rhodamine derivatives Chemosensor Fe3+ Fluorescent probe

a b s t r a c t A new rhodamine B derivative, namely, N1 -(benzo[d]thiazol-2-yl)-N4 -(2-(3 ,6 -bis (diethylamino)-3oxospiro[isoindoline-1,9 -xanthen]-2-yl)ethyl)malea-mide (RDBSF), was designed, synthesized, and structurally characterized to develop a new chemosensor. UV–vis absorption and fluorescence spectroscopic studies show that RDBSF exhibits high sensitivity and selectivity toward Fe3+ in the presence of many other metal cations in a Tris-HCl solution (1 mM, pH = 7.4) containing 30% MeCN by forming a 1:1 complex with Fe3+ . The binding association constant (Ka ) of RDBSF for Fe3+ was estimated as 1.52 × 104 M−1 in the solution. The detection limit of Fe3+ by RDBSF was further determined as 11.6 nM. Intracellular imaging applications demonstrated that RDBSF can be used as a fluorescent probe for the detection of Fe3+ in HepG-2 cells. © 2016 Published by Elsevier B.V.

1. Introduction Iron, which is an important component of hemoglobin, myoglobin and a variety of enzymes, is a trace element essential to organisms in the processes of their physiological activities [1], such as muscle contraction, nerve conduction, enzyme synthesis, synthesis of DNA and RNA, transport of oxygen in the blood, proton transfer, regulation of osmotic pressure in cells and acidbase balance [2]. In these processes, it is not only the macro iron that participates but also some trace metal ions. Studies have shown that the deficiency or excess of iron will equally lead to immune suppression [3], reduced intelligence, and decreased antiinfection ability of the organism, and it will affect the body’s thermoregulatory capacity [4], even inducing a variety of diseases, including hemochromatosis [5], abnormal liver function [6], myocardial injury [7], diabetes [8], cancer [9] and osteoporosis [10]. Therefore, real-time monitoring of the iron ion is of great significance for biology and human health, and the highly selective and highly sensitive detection of iron in living cells has attracted wide attention in the medical and biological chemistry fields [11–15]. Currently, atomic absorption spectroscopy (AAS) [16], inductively coupled plasma atomic emission spectrometry (ICP-AES) [17], inductively coupled plasma mass spectrometry (ICP-MS) [18]

∗ Corresponding authors. E-mail addresses: [email protected] (X. Bao), [email protected] (J. Zhu).

and electrochemical methods [19,20] have been used for the detection of iron ions. These methods are highly sensitivity but often require expensive equipment, complex sample pretreatment and professional operators [21]. Compared with traditional methods, Imaging-PAM, a type of noninvasive molecular imaging technology, with simple operation, low cost, good resolution, short response time, and high sensitivity and selectivity, can be used for the insitu detection of a substrate, displaying great utility in applications for detecting the iron ion [22–26]. Rhodamine derivatives with stable structure, good light stability, and high fluorescence quantum yield characteristics [27–29], are ideal materials for the preparation of fluorescence enhancement and ratiometric probes. Rhodamine B derivatives with a spirolactam structure have no fluorescence, but once the identification groups of rhodamine B derivatives complex with specific ions, open-loop amides will excite a strong fluorescence [30–33]. Based on this mechanism, some reports reveal that rhodamine derivative fluorescent probes were used for the detection of iron ions, [13,34–37] but many of them still possess some weaknesses and inadequacies, such as low sensitivity and significant interference from other metal ions (especially from Cr3+ and Cu2+ ) [38–42]. As the paramagnetic nature of Fe3+ is in the 3d orbit, which can lead to fluorescence quenching [43–45], fluorescence imaging can be hampered both for in-situ monitoring and for in vivo applications. In contrast, turn-on fluorescent probes have the advantages of high sensitivity and strong anti-interference, thus, an increasing amount of attention from researchers has focused on the design and synthesis of a new type probe using fluorescence enhancement for the

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residue was dissolved in CH2 Cl2 and then washed with H2 O and brine. The organic layer was dried with MgSO4 . After removing the solvent, flash chromatography (silica gel; MeOH/CH2 Cl2 , 3:97; v/v) of the residue yielded Compound 1 as a pink solid (3.3 g, 85%). 1 H NMR (500 MHz, CDCl , 298 K, TMS) ı (ppm): 7.90 (dd J = 3.0, 3 1 J2 = 3.0 Hz, 1H), 7.46–7.43 (m, 2H), 7.09 (dd, J1 = 3.0, J2 = 3.0 Hz, 1H), 6.43 (d, J = 8.8 Hz, 2H), 6.37 (d, J = 2.5 Hz, 2H), 6.27 (dd, J = 8.9, 2.6 Hz, 2H), 3.38–3.27 (m, 8H), 3.19 (t, J1 = 6.65 Hz, J2 = 6.6 Hz, 2H), 2.41 (t, J1 = 6.65 Hz, J2 = 6.6 Hz, 2H), 1.16 (t, J1 = 7.0 Hz, J2 = 7.1 Hz, 12H) 13 C NMR (126 MHz, CDCl3 , 298 K, TMS) ı (ppm): 168.59, 153.43, 153.26, 148.79, 132.37, 131.20,128.64, 128.01, 123.79, 122.70, 108.13, 105.64, 97.71, 77.38, 77.13, 76.88, 64.91, 44.31, 43.80, 40.73, 12.56. HRMS (M+H)+ found, 485.2925; calcd for C30 H36 N4 O2 , 484.2838. Elemental analysis: C, 73.16; H, 7.27; N, 11.59%, calculated values for C30 H36 N4 O2 : C, 74.35; H, 7.49; N, 11.56%.

Fig. 1. Chemical structure of RDBSF.

detection of iron ions [46–49]. Herein, we designed and synthesized a novel rhodamine B-derivatized fluorescent chemosensor based on the “OFF-ON” principle of rhodamine, named RDBSF (Fig. 1). RDBSF can detect Fe3+ in a Tris-HCl (1 mM, pH = 7.4) solution containing 30% MeCN for use as a turn-on fluorescence sensor with high sensitivity and specific selectivity toward Fe3+ in the presence of many other ions. 2. Experimental 2.1. Materials and general methods All chemicals were purchased from J&K Scientific (Shanghai, China) and were used without further purification. MeCN was HPLC grade, the other solvents were of analytical grade, and doubledistilled water was used throughout the experiments. The salts used in the stock aqueous solutions of ions were NaCl, KCl, CaCl2 , MgCl2 ·6H2 O, FeCl3 ·6H2 O, CuCl2 ·2H2 O, AlCl3 , SnCl2 ·2H2 O, AgNO3 , HgCl2 , CrCl3 ·6H2 O, Ba(NO3 )2 , KF·2H2 O, KI, Na2 S·6H2 O, Na2 SO4 , K3 PO4 ·2H2 O, NaNO3 , and K2 CO3 . Column chromatography was performed using Haiyang silica gel (type: 200–300 mesh ZCX-2). TLC was performed using a Haiyang silica gel F254 plate. 2.2. General instrumentations The 1 H-(500 MHz) and 13 C NMR (126 MHz) spectra were recorded on a Bruker Avance 500 spectrometer with CDCl3 and DMSO-d6 as solvents and tetramethylsilane (TMS) as an internal standard. High-resolution mass spectra (HRMS) were recorded on a double-focusing high-resolution instrument (Autospec; Micromass Inc.) under electron ionization conditions. All pH measurements were obtained using a PHS- 25C Precision pH/mV Meter (Aolilong, Hangzhou, China) at room temperature (approximately 298 K). Fluorescence measurements were recorded on an Edinburgh FLS920 fluorescence spectrophotometer (Livingston, UK). UV–vis spectra were recorded on a UV-3600 spectrophotometer (Shimadzu, Japan). Cell imaging was performed with an inverted fluorescence microscope (OLYMPUS, IX83). 2.3. Synthesis

2.3.2. Synthesis of Compound 2[50] A solution of Compound 1 (319.62 mg; 0.66 mmol; 1 equiv) and maleic anhydride (97.02 mg; 0.99 mmol; 3 equiv) in CHCl3 (12 mL) was heated to reflux for 20 h, and then, the excess CHCl3 was evaporated in vacuo. The resulting solid was dried under vacuum. The crude product was purified by column chromatography (silica gel; DCM/MeOH, 100/1, v/v) to give Compound 2 (347.11 mg, 90.21%) as a pale white solid. 1 H NMR (500 MHz, CDCl3 , 298 K, TMS) ␦ (ppm): 8.49(s, 1H), 7.90 (d, J = 5.0 Hz,1H), 7.50 (t, J = 1.7 Hz,2H), 7.12 (t, J = 5.0 Hz,1H), 6.40 (d, J = 10.00 Hz 4H), 6.31 (d, J = 15.00 Hz, 3H), 6.20 (s, 1H), 3.38–3.34 (m, 10H), 3.12 (s, 2H), 1.18 (t, J = 7.50 Hz, 12H).13 C NMR (126 MHz, CDCl3 , 298 K, TMS) ı (ppm): 169.98, 165.35, 164.54, 153.09, 152.93, 148.68, 135.69, 132.79, 130.75, 129.74, 128.01, 127.85, 123.65, 122.42, 107.97, 103.73, 97.43, 65.66, 43.96, 41.42, 38.76, 29.23,12.12. HRMS (M+H)+ found, 583.2928; calcd for C34 H39 N4 O5 , 583.2920. Elemental analysis: C, 69.13; H, 6.49; N, 9.33%, calculated values for C34 H38 N4 O5 : C, 70.08; H, 6.57; N, 9.62%. 2.3.3. Synthesis of RDBSF Compound 2 (180 mg, 0.31 mmol), 2-aminobenzothiazole (47 mg, 0.31 mmol), DCC (64 mg, 0.31 mmol), and DMF (3 mL) were placed in a MW monomode reactor in a cylindrical Pyrex vessel. The mixture was irradiated at 135 ◦ C and then dried under vacuum. The residue was dissolved in CH2 Cl2 and then washed with H2 O and brine. The organic layer was dried with MgSO4 . After removing the solvent, flash chromatography (silica gel; MeOH/CH2 Cl2 , 2:98; v/v) of the residue yielded RDBSF as a yellow solid (79.79 mg, 36.12%).1 H NMR (500 MHz, CDCl3 , 298 K, TMS) ␦ (ppm): 15.11(s, 1H), 8.23(s, 1H), 7.91(d, J = 5.00, 1H), 7.90(d, J = 5.00, 1H), 7.84(d, J = 10.00, 1H), 7.79(d, J = 8.00, 1H), 7.49(m, 2H), 7.39(t, J = 10, 1H), 7.27(t, J = 7.58, 1H), 7.12(t, J = 5.00, 1H), 6.43- 6.21(m, 8H), 3.35(m, 10H), 3.19(s, 2H), 1.17(m, J = 7.50,12H). 13 C NMR (126 MHz, CDCl3 , 298 K, TMS) ı (ppm):170.02, 164.35, 161.90, 157.48, 153.20, 152.94, 148.88, 148.66, 134.77, 132.67, 132.04, 130.80, 130.48, 129.85, 127.90, 125.44, 123.62, 123.09, 122.43, 121.91, 120.96, 120.73, 120.49, 118.55, 107.92, 103.86, 97.37, 65.66, 43.95, 41.71, 39.10, 32.74, 31.49, 30.14, 29.90, 29.26, 28.92, 25.49, 22.26, 13.69, 12.16. HRMS (M−H)− found, 713.2919; calcd for C41 H41 N6 O4 S, 713.2910. Elemental analysis: C, 69.00; H, 5.88; N, 10.76%, calculated values for C41 H42 N6 O4 S: C, 68.89; H, 5.92; N, 11.76%. 2.4. Stock solution preparation for spectral detection The stock solutions (50 ␮M) of the metal ions of Cd2+ , Co2+ , Cr3+ , Ag+ , Fe2+ , Fe3+ , Hg2+ , Mg2+ , Mn2+ , Na+ , Al3+ , Ba2+ , Ca2+ ,Cu+ , Cu2+ , Ni2+ , Pb2+ , Sn2+ ,K+ , Li+ ,Zn2+ , F− , Cl− , Br− , I− , HCO3 − , SO4 2− , H2 PO4 − , S2 2− , HPO4 2− , PO4 3− , NO3 − , and CO3 2− and the amino acids Ala (alanine), Arg (arginine), Asn (asparagine), Gly (glycine), Glu (glutamine), Lys (lysine), Met (methionine), Pro (proline), Cys Ir3+ ,

2.3.1. Synthesis of Compound 1[22] To a solution of rhodamine B (3.8 g, 7.95 mmol) in 100 mL of ethanol was added ethane-1,2-diamine (5 mL, 10 equiv). The mixture was refluxed for 12 h and then dried under vacuum. The

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(cysteine), GSH, Ser (serine), Thr (threonine), Leu (leucine), Val (valine), and Hcy (homocysteine) were prepared in MeOH/H2 O (1:2, v/v, pH 7.40). The working solution of RDBSF (10 ␮M) was prepared in MeOH/H2 O (1:2, v/v, pH 7.4). Detection solutions of RDBSF were freshly prepared for spectroscopic measurements. 2.5. UV–vis and fluorescence spectral studies All experiments were performed in a MeOH/H2 O solution (1:2, v/v, pH 7.4, Tris-HCl buffer, 1 mM). In the selectivity experiments, the test samples were prepared by adding an appropriate amount of the ion stock solution or the amino acid solution (50 ␮M) to a 3 mL solution of RDBSF (10 ␮M). In the titration experiment, a 10 ␮M solution of RDBSF was prepared at room temperature with an appropriate amount of the ion stock solution or the GSH solution, which was added to the quartz optical cell using a micropipette. Spectral data were recorded 56 min after the addition of the amino acids or ions. In the fluorescence measurements (slit: 2/2 nm), excitation was provided at 562 nm and emission was at 584 nm; the measurements were collected from 564 to 720 nm.

Fig. 2. The fluorescence spectra of RDBSF (10 ␮M) in solutions of different pH values containing 30% MeCN (␭ex = 562 nm, ␭em = 584 nm, slit: 2/2 nm). Each spectrum was recorded 3 h after the addition of RDBSF.

2.6. Cell studies HepG-2 cells were cultured in Dulbecco’s modified Eagle’s medium (high glucose) supplemented with 10% PBS (fetal bovine serum), 100 units/mL penicillin, and 100 units/mL streptomycin in a 5% CO2 /95% air incubator at 37 ◦ C. HepG-2 cells were seeded at 1 × 105 cells per well in 24-well flat-bottomed plates in an atmosphere of 5% CO2 /95% air at 37 ◦ C and incubated for 2 h before treatment. For living cell imaging, cells were incubated with 10 ␮M RDBSF in culture medium for 2 h in an air incubator at 37 ◦ C. In the control experiment, cells were pretreated with 80 ␮M Fe3+ for 1 h and then incubated with 10 ␮M RDBSF in culture medium for an additional 2 h. Fluorescence imaging was performed after washing the cells with PBS 3 times and recorded using the red channel under an inverted fluorescence microscope. Cytotoxicity of RDBSF was determined by an 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, cells were incubated with different concentrations of RDBSF for 48 h. Cell viability was evaluated by incubating with 0.5 mg/ml (MTT) for 4 h under 5% CO2 /95% air at 37 ◦ C.

Fig. 3. The fluorescence spectra of RDBSF (10 ␮M) with Fe3+ (50 ␮M) in a TrisHCl (1 mM, pH = 7.4) solution containing 30% MeCN(␭ex = 562 nm, ␭em = 584 nm, slit: 2/2 nm). The spectra were recorded at 3 min intervals after the addition of Fe3+ . Inset: trend of fluorescence intensity, with RDBSF recorded at different times.

3. Results and discussion 3.1. Synthesis of RDBSF To obtain Fe3+ -selective fluorescent probes, RDBSF was designed and synthesized in multiple steps in accordance with the route illustrated in Scheme 1. Treatment of rhodamine B with ethylene diamine under the conditions for a condensation reaction afforded Compound 1 according to the reported procedure [22,50]. The product was then grafted to a chain of maleic anhydride molecules to give the key intermediate Compound 2. To obtain the coordination unit for Fe3+ -selectivity, the coupling of Compound 2 and 2-aminobenzothiazole was performed to convert them into RDBSF in 36.12% yield via a condensation reaction. All of the receptors were adequately characterized using 1 H and 13 C NMR and mass spectral methods. NMR spectral changes of ethylene diamine moiety to rhodamine and maleic anhydride moiety to compound 1 had been marked in the attachment section by red rectangle (Supporting materials, Figs. S1–S9). 3.2. Effect of pH values The pH-dependence of RDBSF (10 ␮M) was first evaluated in an optimized MeCN/H2 O (30%) solution, exhibiting a suitable pH range

for RDBSF in biological applications. The fluorescence intensities of the probe were recorded at 3 h after the addition of RDBSF into solutions of different pH values (the pH values were from 2.0 to 12.0) at ␭ex/em = 562/584 nm. As shown in Fig. 2, the results demonstrated that the fluorescence intensity of RDBSF was stable in the pH range of 6.0-10.0, and the fluorescence intensity of the RDBSF-Fe3+ complex can be influenced in the pH range of 4.0-10.0. Hence, the pH range of 6.0-10.0 was reasonable for detecting the fluorescence performance of RDBSF. Based on the above range of reasonable pH values, further fluorescence and UV–vis studies were performed in a MeCN/H2 O (30%, pH, 7.4, Tris- HCl buffer, 1 mM) solution. 3.3. Response time of RDBSF for Fe3+ To evaluate the sensitivity of RDBSF toward Fe3+ , the response time of RDBSF with Fe3+ was estimated in a Tris- HCl solution (1 mM, pH = 7.4) containing 30% MeCN at ␭ex/em = 562/584 nm. The concentration of RDBSF and Fe3+ was 10 ␮m and 50 ␮m, respectively. As shown in Fig. 3, the fluorescence intensity of RDBSF at 562 nm increased rapidly after Fe3+ (50 ␮M) was added to the RDBSF (10 ␮M) solution and stabilized after 69 min following the addition of Fe3+ . These results indicated that RDBSF was a sensitive

Please cite this article in press as: H. Chen, et al., Synthesis and evaluation of a novel rhodamine B-based ‘off-on’ fluorescent chemosensor for the selective determination of Fe3+ ions, Sens. Actuators B: Chem. (2016), http://dx.doi.org/10.1016/j.snb.2016.09.163

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Scheme 1. synthetic route of RDBSF.

sensor for Fe3+ in a Tris- HCl (1 mM, pH = 7.4) solution containing 30% MeCN. Therefore, the reaction time of 69 min was used in subsequent experiments.

3.4. Selectivity studies for metal ions using UV–vis and fluorescence spectra The selectivity of RDBSF (10 ␮M) in a Tris- HCl solution (1 mM, pH = 7.4) containing 30% MeCN was examined with various metal ions (including Na+ , K+ , Ca2+ , Cu+ , Mg2+ , Fe2+ , Fe3+ , Cu2+ , Al3+ , Sn2+ , Ag+ , Hg2+ , Ni2+ , Co2+ , Cd2+ , Cr3+ , Pb2+ , Mn2+ , Li+ , and Ba2+ at 50 ␮M). As shown in Fig. 4, after Fe3+ coordinated with RDBSF, the UV absorption wavelength (␭max = 562) had a slight blue shift, and a highly selective ‘off-on’ absorption was displayed and was significantly stronger than that of all the other metal ions at 558 nm; meanwhile, the effect of Cr3+ and Sn2+ upon the UV absorption changes were negligible at 558 nm (Fig. 4(B)). As also shown in Fig. 4(A), when no Fe3+ ions were added to the solution of RDBSF, the free sample remained colorless, upon the addition of Fe3+ ions to the solution containing RDBSF, the solution changed from colorless to pink. Other various metal ions did not induce any apparent color change after the addition of 5 equiv. of metal ions. The results indicated that RDBSF exhibits excellent selectivity for Fe3+ in a 30% MeCN/H2 O (pH 7.40, Tris-HCl buffer, 1 mM) solution.

Fig. 4. The UV–vis spectra of RDBSF (10 ␮M) with different metal ions (50 ␮M) in Tris- HCl (1 mM, pH = 7.4) containing 30% MeCN. Each spectrum was recorded 69 min after the addition of the metal ions. Insets: (A) color change of RDBSF (10 ␮M) upon addition of different metal ions (5.0 equiv.). (B) Selectivity studies for metal ions using UV–vis from 520 nm to 620 nm.

To achieve a further in-depth analysis of the selectivity of RDBSF for metal ions, the change in the fluorescence intensity upon the

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Fig. 5. The fluorescence spectra of RDBSF (10 ␮M) with different metal ions (50 ␮M) in a Tris- HCl solution (1 mM, pH = 7.4) containing 30% MeCN (␭ex = 562 nm, ␭em = 584 nm, slit: 2/2 nm). Each spectrum was recorded 69 min after the addition of the metal ions.

addition of various metal ions under the same conditions was also investigated by using the fluorescence spectra. As shown in Fig. 5, the fluorescence spectra of RDBSF in a Tris-HCl (1 mM, pH = 7.4) solution containing 30% MeCN displayed a very weak fluorescence at 584 nm (␭ex = 562 nm). When Fe3+ was introduced to the RDBSF solution, a remarkable fluorescence enhancement was observed at 584 nm indicating that the Fe3+ ion induced the formation of the ring-open RDBSF-Fe3+ complex, which exhibited strong fluorescence. All other metal ions showed no particular fluorescence enhancement under the same conditions, except for Cr3+ and Sn2+ , which led to a slight fluorescence enhancement; however, compared with Fe3+ , the influence was almost negligible. These appearances implied that spirolactam ring of RDBSF is open due to Fe3+ -induced delocalization in the xanthene moiety by fluorescence resonance energy transfer [51,52]. This results indicated that RDBSF had better selectivity for Fe3+ than for the other ions. Therefore, the experiments of fluorescence selectivity further demonstrated that RDBSF functions as a highly selective fluorescent chemosensor for Fe3+ . 3.5. Fluorescence titration of RDBSF with Fe3+ To gain further insight into the binding of RDBSF (10 ␮M) with Fe3+ titration against RDBSF in a Tris-HCl solution (1 mM, pH = 7.4) containing 30% MeCN was monitored using fluorescence spectra. As shown in Fig. 6, when no Fe3+ was added to the RDBSF solution, the free RDBSF (10 ␮M) had little fluorescence at 584 nm. However, upon the addition of Fe3+ (0–10 eq.), the titration of Fe3+ with RDBSF led to a significant increase in the emission intensity at 584 nm. The emission intensity reached its maximum value after the addition of 8 equivalents of Fe3+ . The fluorescence quantum yields were calculated to be 0.01 and 0.40 in the absence and presence of Fe3+ ions (8 eq) by using rhodamine B as the standard (ФF = 0.69 in ethanol), respectively [53]. Fe3+ ,

3.6. Detection limit calculations The detection limit of RDBSF for Fe3+ was further calculated based on an experiment with the fluorescence titration (Supporting materials, Fig. S10). The fluorescence spectrum of RDBSF was collected 10 times. Each spectrum was recorded at 10-min intervals at ␭ex/em = 562/584 nm. As shown in Fig. 7, the Fe3+ concentration changed over the range of 0.8–20 ␮M. A good linear regression equation was obtained as y = 5106.25x − 6539.86 (R = 0.9955). The

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Fig. 6. The fluorescence spectra of RDBSF (10 ␮M) for different concentrations of Fe3+ in a Tris-HCl solution (1 mM, pH = 7.4) containing 30% MeCN(␭ex = 562 nm, ␭em = 584 nm, slit: 2/2 nm). Each spectrum was recorded 69 min after the addition of Fe3+ . Inset: plots of fluorescence intensity in which RDBSF was recorded with different concentrations of Fe3+ .

Fig. 7. Detection limit of RDBSF with Fe3+ . The fluorescent spectra of RDBSF (10 uM) with different concentrations of Fe3+ in a Tris-HCl solution (1 mM, pH = 7.4) containing 30% MeCN (␭ex = 562 nm, ␭em = 584 nm, slit: 2/2 nm).

detection limit of Fe3+ was calculated as 11.6 nM by (K = 3) (Supporting Materials, Table S1) [54]. Comparing with some recent reports (Supporting Materials, Tables S2 and S3), the detection limits of RDBSF for Fe3+ show a high detection sensitivity for Fe3+ . 3.7. Calculations of the binding association constant The association constant for RDBSF with Fe3+ was calculated using a Benesi–Hildebrand plot (Supporting Materials, Fig. S11) [55]. The result is shown in Fig. 8. A good linear regression equation was obtained as y = 2.85 × 10−10 x- 4.34 × 10−6 based on the fluorescence titration curves of RDBSF with Fe3+ . Thus, the result of Ka (the binding association constant for Fe3+ ) was 1.52 × 104 M−1 in a Tris-HCl solution (1 mM, pH = 7.4) containing 30% MeCN at ␭ex/em = 562/584 nm, indicating that Fe3+ and RDBSF have a strong binding capacity. 3.8. Job’s plot Next, the complexation ratio of RDBSF with Fe3+ was also investigated in a Tris-HCl solution (1 mM, pH = 7.4) containing 30% MeCN using Job’s plot. The molar concentration of Fe3+ ranged from 0 to

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Fig. 8. The binding association constant of RDBSF with Fe3+ was based on a Benesi–Hildebrand plot, fluorescent spectra (␭ex = 562 nm, ␭em = 584 nm, slit: 2/2 nm).

Fig. 9. Job’s plot of RDBSF in a Tris-HCl solution (1 mM, pH = 7.4) containing 30% MeCN with a total concentration of [RDBSF] + Fe3+ = 50 ␮M. Fluorescent spectra (␭ex = 562 nm, ␭em = 584 nm, slit: 2/2 nm).

1 in a solution of [Fe3+ ]+ [RDBSF]; the total concentration of RDBSF with Fe3+ was 50 ␮M. The results revealed that when the mole fraction of Fe3+ was 0.5, the fluorescence intensity of RDBSF reached the maximum value (Fig. 9), indicating that the ratio at which RDBSF complexed with Fe3+ was 1:1. 3.9. Competitive selectivity of RDBSF for Fe3+ in the presence of other ions To further evaluate the selectivity of RDBSF for Fe3+ , its competitive selectivity for Fe3+ under the same conditions was examined in the presence of other metal cations. As shown in Fig. 10, no significant variation in the emission of RDBSF was observed when comparing the results with or without other metal cations. These results clearly indicated that the other metal cations did not interfere with the detection of Fe3+ . Additionally, the competitive selectivity of RDBSF for Fe3+ under identical conditions was examined in the presence of other anions. As shown in Fig. 11, the emission of RDBSF shows no significant change when introducing various anions, including F− , Cl− , Br− , I− , HCO3 − , SO4 2− , H2 PO4 − , S2 2− , HPO4 2− , PO4 3− , NO3 − and CO3 2− . These results further demonstrate that the presence of anions does not markedly interfere with the detection of Fe3+ ; thus, RDBSF could be used as a highly selective Fe3+ fluorescent chemosensor.

Fig. 10. The competitive selectivity of RDBSF for Fe3+ in the presence of other metal cations was examined with various metal cations (50 ␮M). The selectivity of these metal cations was detected in a Tris- HCl solution (1 mM, pH = 7.4) containing 30% MeCN at ␭ex/em = 562/584 nm. Every spectrum was recorded at 69 min after the addition of Fe3+ (50 ␮M).

Fig. 11. The competitive selectivity of RDBSF for Fe3+ was examined in the presence of various anions (50 ␮M). The selectivity of these anions was detected in a TrisHCl solution (1 mM, pH = 7.4) containing 30% MeCN at ␭ex/em = 562/584 nm. Every spectrum was recorded at 69 min after the addition of Fe 3+ (50 ␮M).

3.10.

1H

NMR study of RDBSF with Fe3+

To determine the reaction mechanism of RDBSF with Fe3+ , the 1 H NMR spectral analysis of RDBSF was studied both in the absence

and in the presence of Fe3+ (Fig. 12). Clearly, compared with RDBSF (3 mg) in a CDCl3 solution (III), all the chemical shifts of RDBSF (3 mg) in a D2 O:CD3 OD (3:1, v/v) showed no changes, except for the proton signals of Hk (II). Upon the addition of 1.0 equiv. of Fe3+ to RDBSF in a CD3 OD:D2 O (3:1, v/v) solution(I), the proton signals of Hg and Hf displayed an apparent downfield shift from the quartet peaks, which were centered at 6.269, 6.293, 6.314 and 6.338 ppm, to triplet peaks, which were centered at 6.635 ppm (␦ = 0.366, 0.342 0.321 and 0.297 ppm, respectively). These changes in chemical shifts can be attributed to the Fe3+ -induced open-loop process of the rhodamine B spirocycle. Next, the proton signals of Hm also displayed apparent blue shifts from the double peaks, which were centered at 6.466 and 6.485 ppm, to single peaks centered at 6.701 ppm (␦ = 0.235 and 0.216 ppm, respectively). Noticeably, the double carbonyl group of the maleic anhydride chain was coupled with Fe3+ . Additionally, the proton signals of Ha displayed apparent red shifts from the triplet peaks at 3.208, 3.219 and 3.232 ppm to another set of triplet peaks at 3.126, 3.139 and 3.150 ppm (␦ = 0.082, 0.080 and 0.082 ppm, respectively). Accord-

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Fig. 12. (I) RDBSF+ 1.0 eq Fe3+ in a D2 O and CD3 OD solution, (II) RDBSF in a D2 O and CD3 OD solution and (III) RDBSF in a CDCl3 solution.

ing to the above results, the mechanism of the interaction of RDBSF and Fe3+ was preliminarily deduced as the complexation of RDBSF and Fe3+ , which is shown in Fig. 12, where Fe3+ might have been complexed with three oxygen atoms on the side chain of RDBSF. In addition, the spirolactam structure of RDBSF was opened, and the fluorescence of RDBSF was excited based on the above coordination effect of RDBSF with Fe3+ . 3.11. Density functional theory (DFT) calculation To further explain the interaction relationship between RDBSF and Fe3+ , the energy-optimized structures of RDBSF as well as RDBSF and Fe3+ were obtained by DFT calculations with the B3LYP method using a suite of Gaussian 09 programs. As shown in Fig. 13, the spatial distributions showed that the spatial structure of RDBSF was a spirolactam and a closed loop structure. However, after adding Fe3+ , the three oxygen atoms on the side chain of the RDBSF-Fe3+ complex and the open-loop amides in the molecular structure of RDBSF emitted strong fluorescence under the coordination function of RDBSF with Fe3+ . To further investigate the sensing mechanism of RDBSF with Fe3+ , the frontier molecular orbitals of RDBSF and the RDBSF-Fe3+ complex are compared in Fig. 13. This comparison indicated that the LUMO of RDBSF was distributed on one of the maleamide chain moieties, and the HOMO

was spread over the group of rhodamine. However, after RDBSF complexed with Fe3+ , the ␲ electrons on the LUMO of RDBSF were distributed over the rhodamine groups, and the HOMO was spread over the 2-aminobenzothiazole group moieties. The energy gaps between the LUMO and HOMO in RDBSF and the RDBSF-Fe3+ complex were calculated as 3.22 and 0.21 eV, respectively. The results show that the binding of Fe3+ to RDBSF to form RDBSF-Fe3+ reduced the energy gap of the HOMO–LUMO of the new product and stabilized the system. 3.12. Cell studies of RDBSF in the presence of Fe3+ Owing to its favorable molecular properties, RDBSF should be suited for fluorescence imaging in living cells. Therefore, we further investigated the applicability of RDBSF as a fluorescent probe for Fe3+ in living HepG-2 cells. Cells treated with only 10 ␮M RDBSF for an additional 2 h. Then the cells were incubated with 80 ␮M Fe3+ for 1 h followed by incubation with RDBSF (10 ␮M) for another 2 h. Comparison with Cells without RDBSF or Fe3+ treatment (Fig. 14A), fluorescence microscopic studies revealed that a lack of fluorescence for human HepG-2 cells after treated with RDBSF alone (Fig. 14B). Upon incubation with Fe3+ , a bright fluorescence was observed within the HepG-2 cells (Fig. 14C). The phase-contrast images in the bright fields of the cells showed that the cells were

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Fig. 13. Molecular orbital diagrams of RDBSF and RDBSF −Fe3+ complex using DFT calculation.

viable during the experiments, which indicates that RDBSF might be cell-permeable to the cells. To evaluate the cytotoxicity of RDBSF, cell viability was determined by an MTT assay in HepG-2 cells with RDBSF concentrations

from 0 to 20 ␮M. Then RDBSF was incubated with HepG-2 cells for 48 h, it showed no toxicity to the cells (Fig. 15). These results suggested that RDBSF may be non-toxic to the cells and suitable to be used as a potential probe for detecting Fe3+ in biological samples.

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Fig. 14. Fluorescence images of HepG2 cells. (A): Cells without RDBSF or Fe3+ treatment; (B): Cells treated with only 10 ␮M RDBSF for an additional 2 h; (C): cells incubated with 80 ␮M Fe3+ for 1 h followed by 10 ␮M RDBSF treatment for an additional 2 h. ((1): bright field, (2):merged images, (3): red channel.). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Job’s plot. The Fe3+ binding ability of RDBSF was further demonstrated using 1 H NMR titration and a DFT calculation, suggesting that the spirolactam structure of RDBSF was opened, and the fluorescence of RDBSF was excited based on the coordination of RDBSF with Fe3+ . Acknowledgements This research was supported by the National Natural Science Foundation of China (81371616), a special appointment of Professor Grant by the JiangSu Province (2015, Prof. Jing Zhu), and the Research Fund for the Doctoral Program of Higher Education of China (20133219120020). We thank the sensor Lab of NUST for providing the supporting facilities for the UV–vis studies.

Fig. 15. Cytotoxicity of RDBSF in human HepG-2 cells. Cells were treated with different concentrations of RDBSF for 48 h and cell viability assay were determined by MTT assay. Data were expressed as means ± SD.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2016.09.163.

4. Conclusion In summary, this study resulted in the development of a RDBSF probe that contains a rhodamine B derivative linked to chain of maleic anhydride and 2-aminobenzothiazole functional groups that undergo complexation with Fe3+ . The chemical structure of RDBSF was analyzed using 1 H NMR, 13 C NMR, and HRMS. The results of performance testing (UV–vis and fluorescence response) show that the RDBSF chemosensor had excellent selectivity and sensitivity for Fe3+ in a Tris-HCl solution (1 mM, pH = 7.4) containing 30% MeCN. The 1:1 complexation mode was achieved based on

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Biographies Hailang Chen graduated from Changzhou University, China, in 2011. He is PhD student in Department of Biochemical Engineering, Nanjing University of Science & Technology. His current research is centered on synthesis of fluorescent probes for cell imaging. Dr. Xiaofeng Bao is an associate professor in Department of Biochemical Engineering, Nanjing University of Science & Technology, China. He received his B.S and M.S. in Chemical Engineering from Nanjing University of Science & Technology in 1999 and 2002. After one year graduate study in the Department of Chemical Engineering, University of Illinois at Chicago, he joined Professor David B. Smithrud’s research group in Department of Chemistry, University of Cincinnati. He received his M.S.

Please cite this article in press as: H. Chen, et al., Synthesis and evaluation of a novel rhodamine B-based ‘off-on’ fluorescent chemosensor for the selective determination of Fe3+ ions, Sens. Actuators B: Chem. (2016), http://dx.doi.org/10.1016/j.snb.2016.09.163

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(Organic Chemistry, 2005) and Ph.D (Organic Chemistry, 2007) from University of Cincinnati. He spent one year (2007–2008) at the Ohio State University Medical Center as a postdoctoral fellow with Professor Villamena Frederick, and additional two-years (2008–2010) at the National Institute of Mental Health, National Institutes of Health as a NIH postdoctoral fellow with Dr. Victor W. Pike. His current research interests include PET tracers for molecular imaging, fluorescent sensors for cell imaging, and rotaxanes for drug delivery agents. Hai Shu is a Master student in Department of Biochemical Engineering, Nanjing University of Science & Technology. Dr. Baojing Zhou is an associate professor in Department of Chemistry, Nanjing University of Science & Technology. He graduated from University of Science and Technology of China in 1998, and received his Ph.D in computational chemistry from University of California-Los Angeles in 2004. He was then trained as a postdoctoral fellow in University of British Columbia (2005–2007), University of Missouri-St. Louis (2007–2009), and University of Calgary (2009–2010). After worked as an assis-

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tant professor in Brandon University for six months, he joined the Department of Chemistry, Nanjing University of Science and Technology as an associate professor in 2011. His current research interests include all fields of computational chemistry. Renlong Ye is Master student in Department of Chemistry, School of Chemical Engineering, Nanjing University of Science & Technology. His current research is centered on computational chemistry. Dr. Jing Zhu is a professor in Department of Pharmacy, Nanjing University of Chinese Medicine. She graduated from Nanjing Normal University in 1999, and received her M.S. (Organic Chemistry, 2006) and Ph.D (Organic Chemistry, 2008) from University of Cincinnati. She spent six years (2008–2014) in Department of Medicine, the Johns Hopkins University as a postdoctoral fellow and additional two months (08/201410/2014) in Columbia University Medical Center as a postdoctoral research scientist. Her current research interests include discovery of novel neuroprotectants for the treatment of neurodegenerative disease, fluorescent sensors for cell imaging, and rotaxanes for drug delivery agents.

Please cite this article in press as: H. Chen, et al., Synthesis and evaluation of a novel rhodamine B-based ‘off-on’ fluorescent chemosensor for the selective determination of Fe3+ ions, Sens. Actuators B: Chem. (2016), http://dx.doi.org/10.1016/j.snb.2016.09.163