A new rhodamine derivative-based chemosensor for highly selective and sensitive determination of Cu2+

Sensors and Actuators B 193 (2014) 679–686

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A new rhodamine derivative-based chemosensor for highly selective and sensitive determination of Cu2+ Premsak Puangploy a , Srung Smanmoo b , Werasak Surareungchai a,c,∗ a School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, Bangkhuntien-chaitalay Road, Thakam, Bangkok 10150, Thailand b Bioresources Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Phaholyothin Road, KlongLuang, Pathumthani 12120, Thailand c Biological Engineering Program, King Mongkut’s University of Technology Thonburi, Pracha-u-tit Road, Toongkru, Bangkok 10150, Thailand

a r t i c l e

i n f o

Article history: Received 4 September 2013 Received in revised form 8 December 2013 Accepted 10 December 2013 Available online 19 December 2013 Keywords: Chemosensor Copper (II) ion Pyrrole Rhodamine derivative Turn-on fluorescence

a b s t r a c t A new rhodamine derivative (R1) has been synthesized by a hydrazone formation of rhodamine B hydrazide with pyrrole-2-carboxaldehyde and its binding affinity to metal ions were examined. R1 shows highly binding selectivity to Cu2+ over commonly coexistent metal ions in neutral aqueous-organic media including alkali, alkaline-earth and transition metals. The linear response to Cu2+ was obtained across the concentration range of 0.4–10 ␮M with the detection limit of 280 nM. Using newly synthesized probe R1, determination of Cu2+ concentration in drinking water and serums, and living cell imaging of Cu2+ were carried out. Also, by incorporating the R1 with filter paper, the sensor was performed in Cu2+ spiked samples. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Copper is an essential trace element in biological systems of plants, living cells, and humans [1–3]. However, at high concentrations, it causes oxidative stress and disorders associated with neurodegenerative diseases for humans, such as Alzheimer’s disease, Wilson’s disease, and Menke’s disease [4]. US-EPA advises the maximum contaminant level goal (MCLG) for copper is 1.3 mg L−1 in drinking water, exceeding the value may cause potential health problem [5]. Several methods have been used to detect metal ions such as atomic absorption spectrometry (AAS) [6,7], inductive coupled mass atomic emission spectrometry (ICP-AES) [8], inductive coupled plasma mass spectroscopy (ICP-MS) [9], plasmon resonance Rayleigh scattering (PRRS) spectroscopy [10], and electrochemical methods [11,12]. However, they are rather complicated by pretreatment procedures, time-consuming analysis, costly instruments, and the fact that they are unable to be used in the field. Fluorescent chemosensors for transition-metal ions have attracted much attention due to their implicitness, high selectivity and sensitivity, low cost, and real-time monitoring

∗ Corresponding author at: School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, Bangkhuntien-chaitalay Road, Thakam, Bangkok 10150, Thailand. Tel.: +66 2 470 7474; fax: +66 2 452 3455. E-mail address: [email protected] (W. Surareungchai). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.12.037

[13]. For the determination of Cu2+ , almost previous works have reported the “on-off” fluorescence probes [14–17]. This was due to its paramagneticity of the Cu2+ ion (3d9 ), leading to the fluorescence quenching of the Cu2+ -probe complex. The quenching behavior of the probe, however, is not as sensitive as a fluorescence enhancement response in terms of signal detection. Therefore, the development of a turn-on fluorescence probe is of interest. Rhodamine fluorophore and its derivatives have been extensively studied as turn-on type chemosensors due to their excellent photophysical properties such as the long absorption and emission wavelength, high fluorescence quantum yield, large extinction coefficient, and high photostability [18]. The rhodamine derivatives with an opened spirolactum ring (effecting from metal ion chelation) give off a strong fluorescent emission and pink color [19]. Based on this mechanism, fluorescent enhancement of rhodamine probes have been reported for Cu2+ detection [20–24]. However, these chemosensors are disadvantageous from other metal ions interferences, long response times, and complicated procedures for synthesis [25–30]. Recently, the furan-bound rhodamine derivative was reported as Cu2+ selective chemosensor with high selectivity of Cu2+ in living cells [31]. However, this furan derivative showed the low binding affinity with Cu2+ and the interference effect of Cu2+ with Hg2+ . Instead of furan, we are interested to use pyrrole as a linker because N atom of pyrrole makes high electrostatic contributions to the binding of transition metal ions better than O atom of furan [32]. Also, Cu2+ is able to chelate to form a bidentate

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Scheme 1. Synthesis of R1.

molecular structure via carbonyl O atom of the xanthenes group and N atom of pyrrole; expectedly a better selectivity toward Cu2+ . Herein, we designed and synthesized a new rhodamine derivative bearing a pyrrole unit as a fluorogenic and chromogenic sensor for Cu2+ . Among the various metal ions, this probe exhibits remarkably enhanced absorbance intensity and shows significant turn-on fluorescence intensity for Cu2+ in HEPES/acetonitrile buffer at physiological condition. In addition, the probe has been successfully applied for rapid and sensitive measuring for Cu2+ in drinking water, human serum samples and in vivo imaging of HeLa cells. In addition, the probe was demonstrated in a form of paper-based sensor for applying in real samples. 2. Experimental 2.1. Reagents and instruments Rhodamine B, hydrazide hydrate, pyrrole-2-carboxaldehyde, and 2-[4-(2-hydroxyehtyl) piperazin-1-yl] ethanesulfonic acid (HEPES) were purchased from Sigma–Aldrich. Acetonitrile (Carlo Erba) and double-distilled water were used throughout the experiment. Solutions of Ba2+ , Ca2+ , Co2+ , Cu2+ , Fe2+ , Fe3+ , Hg2+ , K+ , Mg2+ , Mn2+ , Na+ and Ni2+ were prepared from their chloride salts and solutions of Ag+ , Cd2+ , Cr6+ , Fe2+ , Pb2+ and Zn2+ were prepared from their nitrate salts. Thin layer chromatography (TLC) was carried out using silica gel 60 F254 (Merck) and column chromatography was conducted over silica gel 60. NMR spectra were recorded on a Bruker 400 MHz spectrophotometer. Mass spectra were obtained on a Bruker microTOF LC-ESI spectrometer. Absorption spectra were measured on a GCB Cintra 404 UV-Vis spectrometer. Fluorescence spectra measurements were recorded on a JASCO FP-6500 spectrofluorometer. All measurements were operated at a room temperature of ∼298 K.

CDCl3 ), ı (ppm): 164.52, 152.49, 151.63, 148.48, 138.49, 132.69, 128.55, 128.31, 127.75, 127.51, 123.16, 122.70, 120.71, 113.32, 109.02, 107.62, 105.29, 97.40, 65.37, 43.88, 12.19. HRMS m/z calcd. for C33 H35 N5 O2 : 533.2791 found: 534.2858 [R1+H]+ . 2.3. Measurement procedures 2.3.1. General procedure for Cu2+ determination A 1 mM stock solution of R1 was prepared by dissolving R1 in absolute acetonitrile. A standard stock solution of Cu2+ ion (10 mM) was prepared by dissolving an appropriate amount of copper chloride in Milli-Q water. The complex solution of Cu2+ /R1 was prepared by adding 100 ␮L of stock solution of R1 and 100 ␮L of the stock solution of Cu2+ in a volumetric flask (10 mL). After adjusting the final volume with acetonitrile/HEPES buffer (10 mM, pH 7.0, 1:1 of v/v), the solution was placed at room temperature for 30 min. After that, the solution was measured with fluorescence spectroscopy. The excitation wavelength was performed at 510 nm. Both excitation and emission slit widths were 3 nm. All solutions were protected from light and kept at 4 ◦ C for further use. A blank solution of R1 was prepared under the same conditions without Cu2+ . Stock solutions of other metal ions were prepared in water with a similar procedure. 2.3.2. Detection of Cu2+ in human serums The human serum samples were treated with 0.05% HNO3 . After that, the serum samples were centrifuged and the protein components were removed. The supernatant solution was collected and kept at 4 ◦ C. Next, the supernatant serum was transferred and mixed with R1 solution in a 10 mL volumetric flask. Acetonitrile/HEPES buffer (10 mM, pH 7.0, 1:1 of v/v) was added to adjust to the final volume of 10 mL. The solution was left at room temperature for 30 min before measuring with a fluorescence spectrophotometer with 510 nm of excitation wavelength.

2.2. Synthesis of R1 Rhodamine B hydrazide was prepared according to the literature [33]. Rhodamine B hydrazide (1.0 mmol, 0.4560 g) and pyrrole-2carboxaldehyde (1.2 mmol, 0.1141 g) were mixed in boiling ethanol with the addition of 3 drops of acetic acid (Scheme 1). After 24 h of stirring, the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was cooled down to room temperature and poured into a brine solution before extracting with dichloromethane. The combined organic extracts were dried over anhydrous sodium sulfate before being filtered and the solvent was completely evaporated. The crude solid product was further purified by silica gel column chromatography [hexane/ethyl acetate (3/2, v/v)] before the desired R1 was obtained as a colorless solid in 45% yield. 1 H NMR (400 MHz, CDCl3 ), ı (ppm): 8.40 (s, 1H), 7.92–7.99 (d, J = 7.1 Hz, 1H), 7.40–7.48 (m, 2H), 7.08–7.12 (d, J = 7.0 Hz, 1H), 6.70 (s, 1H), 6.49–6.53 (d, J = 9.0 Hz, 2H), 6.39–6.42 (d, J = 2.0 Hz, 2H), 6.22–6.28 (m, 3H), 6.01–6.03 (m, 1H), 3.37–3.48 (q, J = 7.0 Hz, 8H), 1.15–2.01 (t, J = 7.0 Hz, 12H). 13 C NMR (400 MHz,

2.3.3. Cell culturing and imaging Living HeLa cells (cervical cancer cells) were provided by the National Center for Genetic Engineering and Biotechnology (Thailand). Cells were grown in EMEM supplemented with 10% FBS (fetal bovine serum), 2 mM l-glutamine, 1 mM sodium pyruvate, 0.1 mM essential amino acids and 1.5 g/L sodium bicarbonate in an atmosphere of 5% CO2 and 95% air at 37 ◦ C. Cells were seeded on a 96-well plate at 5 × 105 cells per well and allowed to adhere for 24 h. Immediately before the experiments, the cells were washed with phosphate-buffered saline (PBS) three times and then incubated with 20 ␮M of R1 (in the culture medium) for 20 min at 37 ◦ C. The R1 probe was taken up into cells by endocytosis mechanism [34,35]. After washing with PBS (three times) to remove the remaining R1, the treated cells were incubated with 50 ␮M CuCl2 (in the culture medium) for 20 min at 37 ◦ C. The remaining copper ions were removed by washing with PBS three times before adding 200 ␮L of PBS into the well. Fluorescent imaging was performed with an Olympus IX71 inverted fluorescence microscope to record

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Fig. 1. The optical changes of R1 (10 ␮M) in the presence of various metal ions (100 ␮M of each) in acetonitrile/HEPES buffer (10 mM, pH 7.0, 1:1 of v/v). (a) The photos of the color (top) and fluorescent (bottom) changes of R1 with other metal ions. (b) Absorbance and (c) fluorescence intensity of R1 upon addition of different metal ions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

the images before and after the addition of Cu2+ ions into the cells. The emission wavelength was collected from 510 to 550 nm. 2.3.4. Cytotoxicity assay The green fluorescent protein (GFP) assay was performed to evaluate the cytotoxicity effect of R1 [36]. GFP assay carried out by adding 45 ␮l of Velo cells suspension at 3.3 × 104 cells/ml to each well of 384-well plates containing 5 ␮l of R1 (final concentration equal to 20 ␮M) previously diluted in 0.5% DMSO, and then incubating for 4 days in 37 ◦ C incubator with 5% CO2 . Fluorescence signals are measured by using SpectraMax M5 microplate reader (molecular devices) in the bottom-reading mode with excitation and emission wavelengths of 485 and 535 nm. 2.3.5. Preparation of R1 paper based sensor and sample tests R1 was dissolved with methanol in which the filter papers were also added and incubated for 30 min. The collected papers were dried in a dark desiccator under vacuum for 24 h. The R1 sensor was tested by dipping in Milli-Q water at different concentrations of Cu2+ spiked, also river samples that were collected from Connecticut River at Amherst town, MA were evaluated. To observe the color change the sensor was allowed drying in air for 1 min. 3. Results and discussion 3.1. Selectivity R1 was synthesized by the condensation reaction between rhodamine B hydrazide and pyrrole-2-carboxaldehyde as detailed in Section 2. Its structure was investigated by mass spectra and 1 H NMR and 13 C NMR spectra (see Figs. S1, S2, and S3, respectively, in the supporting information). R1 shows colorless or non-fluorescent solutions in equal ratios (v/v) of 10 mM HEPES buffer (pH 7.0) and acetonitrile, indicating that the closed spirolactam form is predominant. The observed result corresponds to the distinctive spirocycle carbon that appeared at 65.37 ppm in the 13 C NMR spectrum (see Fig. S3 in the Supporting information).

In Fig. 1(a), a change in color and fluorescence in the presence of Cu2+ was evidently exhibited, whereas other metals showed neither a color nor a fluorescence change. Upon the addition of Cu2+ , therefore, the color of R1 abruptly changed from colorless to pink indicating that the equilibrium shifted to the rhodamine spirolactam ring-opened form. The shift in R1 equilibrium is also associated with the fluorescent enhancement. Confirmatively, by absorbance spectra only Cu2+ could give a strong absorbance around 555 nm together with a shoulder peak of ∼520 nm (Fig. 1b). Also, the fluorescence change upon the addition of Cu2+ (100 ␮M) occurred in which the fluorescence enhancement peak at 575 nm increased due to the opening of the rhodamine ring (Fig. 1c). The presence of alkali- and alkaline-earth metals, such as Na+ , K+ , Mg2+ , Ca2+ , and Ba2+ (100 ␮M of each) and transition metals such as Ag+ , Cd2+ , Co2+ , Cr6+ , Fe2+ , Fe3+ , Hg2+ , Mn2+ , Ni2+ , Pb2+ , and Zn2+ (100 ␮M of each) did not induce significant absorbance and fluorescent changes. Therefore, the results indicate that R1 gives an excellent selectivity for Cu2+ ions in both fluorimetric and colorimetric changes. 3.2. Optimization Experimental conditions including solvent media, pH, and response time were optimized. Since R1 has a polarity of an N atom in a pyrrole ring and a non-polarity of conjugated bonds in a xanthenes ring, organic solvents and water content affected the coordination reaction. Therefore, in this study, binary watersolvents were investigated by a ratio of 1:1 of acetonitrile, methanol, or dichloromethane (either one of these) and 10 mM HEPES buffer (pH 7.0). R1 (10 ␮M) binding with Cu2+ (10 ␮M) was excited at 510 nm and the fluorescent emission intensity was recorded at 575 nm. Results show that in an acetonitrile–water solution, high fluorescence intensities (ratio between R1 with and without Cu2+ ) were obtained (see Fig. S4 in the Supporting information). Therefore, acetonitrile was then chosen as a proper solvent for further experimentation. Varying the ratio of acetonitrile in 10 mM HEPES buffer (pH 7.0) was also investigated. The fluorescence signal increased when the acetonitrile proportion increased up to 50% (Fig. S5 in the

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Fig. 3. Fluorescent intensity of R1 (10 ␮M) in the presence of various concentrations of Cu2+ (0.4–10 ␮M) in 10 mM HEPES/acetonitrile (1:1, v/v, pH 7.0). Excitation wavelength was 510 nm. Fig. 2. Fluorescence intensity of R1 (10 ␮M) () and R1 + Cu2+ () in 10 mM HEPES/acetonitrile (1:1, v/v, pH 7.0) as a function of different pH values (ex = 510 nm).

Supporting information). The increasing proportion of organic solvents supports the binding of R1 and Cu2+ . However, the overloading proportion of organic solvents can cause low solubility of Cu2+ in aqueous medium and causes low affinity between R1 and Cu2+ [18]. With the acetonitrile proportion higher than 50%, the signal significantly decreases. Since acetonitrile gave the highest fluorescent intensity of R1 at 50%, this ratio was chosen as the optimal condition and used for the subsequent experiments. Fig. 2 shows that the fluorescence responses obtained from R1 and binding R1 with Cu2+ depended on the pH. For a low pH (pH <5), the fluorescence signals of both R1 and R1+Cu2+ were high but not significantly different compared to R1 with and without Cu2+ . The result implies that at low pH, the protonized states of spirolactam formed in both the R1 and R1+Cu2+ complex. Whereas, at a pH of 6–12, the fluorescence signal of R1 was low and almost stable, suggesting that they are likely to be spiroring-closing forms. The presence of Cu2+ , at a pH of 6–7 caused signal enhancement since Cu2+ interacted with R1 and the rhodamine ring opened. Also, the solution turned from colorless to pink which was observed by the naked eye and from a non-fluorescent to a fluorescent orange color under UV light. The pH value that gave the highest different

fluorescence signal of R1 and R1+Cu2+ was pH 7.0. Therefore, this pH value was chosen as the optimum experimental condition. 3.3. Sensing characteristics By the above-described optimum conditions, the fluorescence signal is linearly proportional to the concentration of Cu2+ within the range of 0.4–10 ␮mol L−1 (R2 = 0.991) (Fig. 3). The limit of detection (LOD) was found to be 0.28 ␮M based on the 3S/N method. The relative standard deviation for five repeated measurements of 10 ␮mol L−1 Cu2+ was 6.75%. The fluorescence responses of the R1 probe to various cations and its selectivity for Cu2+ were investigated. The result is shown in Fig. 4. The black bar portion describes the fluorescence response of R1 (10 ␮M) to different metal ions of interest. It obviously found no significant fluorescence intensity changes in the present various metal ions including Ag+ , Fe3+ , Pb2+ , Zn2+ (10 ␮M), Cd2+ , Hg2+ , Mn2+ (50 ␮M), Ba2+ , Ca2+ , Co2+ , Cr6+ , Mg2+ , Ni2+ (100 ␮M), K+ , and Na+ (500 ␮M). R1 only enhanced the fluorescence intensity with the presence of Cu2+ (10 ␮M). It is inferred that the proposed probe reveals high selectivity to Cu2+ over other metal ions. The response time of R1 with Cu2+ was investigated by monitoring the fluorescent emission intensity of R1 (10 ␮M) reacting with Cu2+ (10 ␮M) in acetonitrile/10 mM HEPES buffer (1:1, v/v,

Fig. 4. Fluorescence intensity of 10 ␮M R1 upon the addition of various metal ions in 10 mM HEPES/acetonitrile (1/1, v/v, pH 7.0). Black bars represent the fluorescence response of R1 to the metal ion of interest (10 ␮M of Cu2+ , Ag+ , Fe3+ , Pb2+ , Zn2+ , 50 ␮M of Cd2+ , Hg2+ , Mn2+ , 100 ␮M of Ba2+ , Ca2+ , Co2+ , Cr6+ , Mg2+ , Ni2+ , 500 ␮M of K+ , and Na+ ). Gray bars represent the fluorescence response of R1 in the presence of the Cu2+ ion (10 ␮M) and corresponding metal ions (above concentrations). Excitation wavelength = 510 nm.

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Fig. 5. Job plot of the R1–Cu2+ complex in 10 mM HEPES buffer/acetonitrile (1:1, v/v, pH 7.0). The total concentration of R1 and Cu2+ was 20 ␮M. Excitation wavelength was performed at 510 nm.

pH 7.0) at an excitation wavelength of 510 nm (Fig. S6 in the Supporting information). The fluorescence intensity was immediately increased and stable within 2 min, which was a significantly faster analysis time for Cu2+ detection in comparison to the rhodamine B hydroxylamide probe (response time took 2 h) [26]. 3.4. Binding capability The job plot method was used to determine the binding stoichiometry of the R1–Cu2+ complex. The total concentrations were kept at 20 ␮M. The molar ratio was given by [Cu2+ ]/([Cu2+ ]+[R1]) and measured from 0 to 1. As seen in Fig. 5, the maximum value of the molar ratio was 0.5. This means that the molar ratio of the R1–Cu2+ complex was 1:1 in the binding stoichiometry. To examine the binding constant (Ka ), fluorescent titration of spectra of R1 (10 ␮M) in the presence of various concentrations of Cu2+ (0–90 ␮M) was conducted. When Cu2+ increased, the fluorescence intensity of the R1–Cu2+ complex increased, as seen in Fig. 6. It should be noted that at more than 70 ␮M of Cu2+ , the fluorescence intensity reached the saturated complex. We estimated Ka using the 1:1 binding mode of the Benesi–Hildebrand correlation [31]: IF0 (IF − IF0 )

=

1 f



1 +1 Ka [M]

(1)

where IF0 is the fluorescence intensity of R1 at 575 nm. IF is the fluorescence intensity of R1 at 575 nm upon the addition of different concentrations of Cu2+ and f is the fraction of the initial fluorescence which is accessible to the sensor. [M] is the concentration of Cu2+ . The resulting Benesi–Hildebrand plot, shown in the inset of Fig. 6, had a Ka of 1.18 × 104 M−1 . The Ka obtained illustrates a strong binding ability of Cu2+ with the imine-rhodamine probe, compared to the monoboronic acid-conjugated rhodamine probe

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Fig. 6. Fluorescent titration spectra of R1 (10 ␮M) in the presence of various concentrations of Cu2+ ion (0, 0.4, 0.6, 0.8, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, and 90 ␮M) The inset shows the Benesi–Hildebrand plot of the R1–Cu2+ complex to indicate a 1:1 R1–Cu2+ ratio. These spectra were measured in 10 mM HEPES buffer/acetonitrile (1:1, v/v, pH 7.0). Excitation wavelength was 510 nm.

(2.8 × 103 M−1 ) [21], the naphthalimide–rhodamine fluorescent probe (5.2 × 103 M−1 ) [22], or the furan–rhodamine fluorogenic probe (8.9 × 103 M−1 ) [31]. For studying the reversible property of the probe, excess EDTA solution was added into the mixture solution of R1 (20 ␮M) and Cu2+ (20 ␮M). The solution color changed from pink to colorless and the fluorescence intensity was almost quenched, as shown in Fig. S7 in the Supporting information. This means that EDTA can chelate to Cu2+ by replacing R1. However, after adding excess Cu2+ to the system again, the solution regenerated the fluorescent signal. Therefore, the R1 sensor has the ability to be a reversible chemosensor for Cu2+ . 3.5. Complexation mechanism The complexation mechanism of R1 with Cu2+ is based on the spiroring-opening mechanism, which is the same as the spirocycle rhodamine derivatives [37–40]. In the absence of Cu2+ , the solution of R1 is colorless and non-fluorescent because R1 exists in the closed spirolactam form. However, upon the addition of Cu2+ into the R1 solution, the ring of spirolactam R1 opened, resulting in color changes and fluorescence enhancement. The data from the EDTA experiment can be used to support this reversible spirocycleopening mechanism. To determine the complexation between R1 and Cu2+ , the partial infrared spectra of R1 and R1–Cu2+ are shown in Fig. S8 in Supporting information. For compound R1, the 1614 and 1738 cm−1 bands correspond to the conjugated C O group of spirolactam and the amide carbonyl group, respectively. While in the case of R1+Cu2+ ,

Scheme 2. The proposed complexation mechanism of R1 with Cu2+ .

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Fig. 8. Colorimetric detection by R1 (1 × 10−3 M) sensor: (a) in Cu2+ spiked Milli-Q water at (1) 0 M, (2) 10−7 M, (3) 10−6 M, (4) 10−5 M, (5) 10−4 M, (6) 10−3 M, and (7) 10−2 M; and (b) in Cu2+ spiked natural water at (1) 10−6 M and 10−5 M. Fig. 7. Energy-minimized structure for the R1–Cu2+ complex by DFT calculation.

the peak at 1614 cm−1 shifted to 1443 cm−1 which indicated that the carbonyl group of spirolactam was involved in Cu2+ coordination. In addition, the infrared spectra of R1–Cu2+ also showed an absent of the amide C O frequency from 1738 cm−1 , which might be present all enolated form of R1 with binding of Cu2+ complexation. To further clarify the coordination behavior of the R1–Cu2+ complex, we performed density functional theory (DFT) calculations with the Becke-3-Lee–Yang–Parr (B3LYP) exchange function using the Gaussian 03 package. Fig. 7 represents the molecular geometry optimization according to the 1:1 binding stoichiometry of R1 with the Cu2+ ion. The Cu atom is introduced by the electronwithdrawing carbonyl group. Hence, non-bonding electrons of the N atom of the xanthenes group have delocalized in the whole

␲-orbital of R1. The carbonyl O atom has the strong ability to coor˚ At dinate Cu2+ , which corresponds to the bond length (1.8345 A). the same time, the electron-donating N atom of a pyrrole ring has ˚ high affinity to bind to Cu2+ with short bond lengths (1.8135 A). Considering their bond lengths, we propose that the Cu2+ atom can coordinate with carbonyl O and pyrrole N atoms in a bidentate molecular structure, as illustrated in Scheme 2. 3.6. Applications R1 was employed as a fluorescent sensor for the determination of Cu2+ in drinking water and human serums. Cu2+ free samples were spiked with standard Cu2+ solution. Results were satisfactory, agreeing with the atomic absorption spectroscopic (AAS) method as shown in Table 1. The percentages of relative error were less than 10%. To further realize the practical application, the R1 as

Fig. 9. Confocal fluorescence and bright-field images of HeLa cells. (a) Fluorescence image of HeLa cells incubated with 20 ␮M R1 for 20 min. (b) Fluorescence image of HeLa cells incubated with 20 ␮M R1 for 20 min and then treated with 50 ␮M Cu2+ for 20 min. (c) Bright-field transmission image of cells shown in panel (b). The overlay image of (b) and (c) is shown in (d).

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Table 1 Determination of Cu2+ in drinking water and human serum samples. Samples

Cu2+ spiked (mol L−1 )

Drinking water

−6

5 × 10 10 × 10−6

Human serum

5 × 10−6 10 × 10−6

a b

AAS methoda (mol L−1 )

Proposed methodb (mol L−1 )

Relative error (%)

(5.16 ± 0.18) × 10 (10.75 ± 0.14) × 10−6

(4.86 ± 0.02) × 10−6 (10.02 ± 0.16) × 10−6

6.11 7.30

(5.35 ± 0.38) × 10−6 (10.54 ± 0.30) × 10−6

(4.90 ± 0.22) × 10−6 (9.86 ± 0.11) × 10−6

9.08 6.85

−6

Measurement by atomic absorption spectroscopy. Average of three recipicates by the proposed method.

paper-based sensors were prepared and evaluated its applicability to detect Cu2+ in water samples. The dipping time of R1 sensor into a sample solution only takes a min for coloring (varying dipping time reported in Fig. S9 in the Supporting information). Fig. 8a exhibits the color changes of the sensor in Cu2+ spiked Milli-Q water at different concentrations. We can observe the color at the lowest concentration of 10 ␮M. The R1 sensor was also applied to samples from the river. Result is illustrated in Fig. 8b. When 10 ␮M Cu2+ was spiked, the color obviously appeared. For further use in cell imaging applications, HeLa cells were tested by inverted fluorescence microscopy before and after incubation of Cu2+ in the cells. The result is shown in Fig. 9. Cells that were incubated with 20 ␮M R1 for 20 min at 37 ◦ C showed a very weak intracellular fluorescence (Fig. 9a). The cells were washed with PBS three times and treated with 50 ␮M Cu2+ for 20 min at 37 ◦ C. The fluorescent image of cells increased significantly (Fig. 9b). Bright-field measurement after the treatment with Cu2+ and R1 showed the cell growth by the imaging experiment in Fig. 9c. The overlay image of b and c is shown in Fig. 9d. It indicates that the distributions of Cu2+ ions in cells were observed by the fluorescence signal in the perinuclear area of the cytosol. Thus, R1 could be used as a fluorescent probe for detecting Cu2+ in living cells. To evaluate the cytotoxicity of R1, the probe R1 was diluted in 0.5% DMSO with Velo cells suspension in 384-well plates, and then incubated for 4 days in 37 ◦ C with 5% CO2 . The results showed that the cellular ability still remained 63.8% after treatment with 20 ␮M of R1. It means R1 gave low toxicity to cultured cell and can be applied for monitoring Cu2+ in living cells.

4. Conclusion In summary, synthesized rhodamine B hydrazide pyrrole showed a high selectivity and sensitivity to Cu2+ . The interaction of R1 and Cu2+ was reversible with 1:1 binding stoichiometry in acetonitrile–water medium (1:1 of v/v). The detection limit was found to be 280 nM, which is sufficient for real applications in drinking water and human serums. The probe has also been successfully applied for bio-imaging of live HeLa cells. R1 paper based sensor was performed by naked eye inspection without spectroscopic instruments.

Acknowledgments P.P. acknowledges a Ph.D. scholarship from Thailand’s Office of the Higher Education Commission. Human serum samples were obtained from Dr. Kulachart Jangpatarapongsa at Mahidol University. Thanks to Dr. Nakorn Niamnont at KMUTT for the invaluable discussion. We also appreciate Rotello’s lab at UMASS for supporting paper-based sensors preparation. This work is supported by the Higher Education Research Promotion and National Research University Project of Thailand (Grant no. 55000667).

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Biographies Premsak Puangploy is a Ph.D. candidate at King Mongut’s University of Technology Thonburi. He is interested in the synthesis and characterization of optical chemosensor for biological and environmental detection. Srung Smanmoo was obtained a Ph.D. degree from Sheffield University, England. Currently, he is a researcher in Bioresources Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC). His research interests are supramolecular chemistry and chemosensor for biological and environmental analysis. Werasak Surareungchai is an associate professor at King Mongkut’s University of Technology Thonburi. His researches focus on electroanalytical chemistry and bionanotechnology for medical, biological, and environmental analysis.