A water compatible turn ‘on’ optical probe for Cu2+ based on a fluorescein–sugar conjugate

Sensors and Actuators B 196 (2014) 345–351

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A water compatible turn ‘on’ optical probe for Cu2+ based on a fluorescein–sugar conjugate Uzra Diwan a , Ajit Kumar a , Virendra Kumar a , K.K. Upadhyay a,∗ , P.K. Roychowdhury b a b

Department of Chemistry (Centre of Advanced Study), Faculty of Science, Banaras Hindu University, Varanasi 221 005, India Analytical Division, Chembiotek Research International, Kolkata 700 091, India

a r t i c l e

i n f o

Article history: Received 26 December 2013 Received in revised form 6 February 2014 Accepted 8 February 2014 Available online 17 February 2014 Keywords: Galactose Fluorescein Optical sensor Fluorescence and Cu2+ ion

a b s t r a c t A fluorescein–sugar conjugated chromo-fluorogenic turn ‘on’ probe (FG) has been synthesized for detection of Cu2+ . The FG comprises of fluorescein as an efficient fluorophore and a sugar moiety, viz., galactose as the binding unit. The inclusion of galactose into FG led towards its good water compatibility. When Cu2+ was added in 70% aqueous HEPES buffered solution (pH 7.4) of FG, the absorbance and the fluorescence spectral pattern of the same were modulated dramatically with observation of absorption and emission bands at 632 and 515 nm, respectively. The detection limit from fluorescence titration was calculated as 6.32 nM which further establishes high sensitivity of FG towards Cu2+ . The spectral studies for the interaction of FG with Cu2+ indicated towards metal ion triggered spirolactam ring opening of FG as the mechanistic pathway of the sensing phenomenon. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The modulation of absorption and/or fluorescent spectra of many organic molecules upon binding with a specific analyte are very important and is an effective way to detect a particular analyte quantitatively and qualitatively both [1]. The optical sensors are being employed in applications ranging from clinical toxicology, environmental, bioinorganic chemistry, bioremediation, and waste management, etc. [2]. The Cu2+ is the third most abundant divalent metal ion in the human body, after Fe2+ and Zn2+ and is an essential trace element in biological systems [3]. The same also plays a crucial role in a variety of fundamental metabolic processes in organisms ranging from bacteria to mammals [4]. Nevertheless, while a lowlevel background intake of copper is indispensable while high doses of copper can be harmful and even toxic to biological systems [5,6]. Excess copper (II) can cause serious health problems [6]. Due to widespread use of Cu2+ the same is a significant metal pollutant [7] also. Till date, a number of technologies have been developed to detect Cu2+ but the need of sophisticated instrumentations and highly trained operators are major bottle necks in their routine applications [8]. In last couple of decades optical sensors i.e., chromogenic/fluorogenic sensors have come up as the cheapest and convenient options for the detection of analytes at their very

∗ Corresponding author. Tel.: +91 542 670 2488. E-mail addresses: [email protected], [email protected] (K.K. Upadhyay). http://dx.doi.org/10.1016/j.snb.2014.02.031 0925-4005/© 2014 Elsevier B.V. All rights reserved.

low concentrations [9]. The colorimetric response for Cu2+ are quite common and have been observed with a number of sensors even at its low level [10,11] but the turn ‘on’ fluorescent response for the same are rare due to its paramagnetic nature which ultimately acts as fluorescent quenchers rendering low signal output [11]. To get the turn ‘on’ probe for Cu2+ a number of moieties and their derivatives have been explored by various workers and many successful fluorescent turn ‘on’ chemosensors for Cu2+ have been developed [12]. But most of them have one or other problems in their practical applications, such as their water solubility, poor response time, and poor selectivity [8]. Therefore, we thought it worthwhile to develop a new fluorescent turn ‘on’ probe for Cu2+ having high sensitivity, selectivity with high water compatibility. In this context our literature survey revealed that the rhodamine and fluorescein derivatives are able to give turn ‘on’ response for various metal ions including Cu2+ in visible to NIR region [13]. The cyclic forms of these dyes are non-fluorescent while their spirolactam ring-opening upon binding with a particular metal ion finally result into a strong fluorescence and obvious colour change [13]. Due to their large visible-range extinction coefficients and high fluorescence quantum yields, these dyes are excellent choice as efficient chromo-fluorophores [14]. In recent years, several rhodamine-based chemosensors and chemodosimeters for metal ions have been studied [13,14] but fluorescein-based probes have received comparatively little attention hitherto. Fluorescein has also been used as a tracer in various clinical studies such as angiography, reflecting its low toxicity [15].

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solvent effect towards some metal ions; (b) upon coordination of Cu2+ , the spirolactam of fluorescein moiety got opened, producing obvious colour and optical changes selectively for Cu2+ . The detection limit of FG towards Cu2+ was found to be 6.32 × 10−9 M which is highest among the earlier carbohydrate based Cu2+ fluorescent sensor and is comparable to other excellent Cu2+ selective sensors reported in literature so far [21,23,25]. 2. Experimental 2.1. Synthesis of FG

Scheme 1. Chemical structure of FG.

Thus, we thought it worthwhile to have fluorescein as a constituent of our next design of a chemosensor for Cu2+ . A variety of aromatic aldehydes were condensed with fluorescein hydrazide to get efficient optical chemosensors for a number of metal ions by various workers till date [14a,16]. This time we tried to incorporate the sugar moiety having an aldehyde group like galactose for the first time with fluorescein to have an efficient and a water compatible probe. Our this strategy led the synthesis of a new fluorescein-galactose conjugated optical probe (FG) for Cu2+ where fluorescein works as fluorophore while the galactose acted as a metal binder as well as served the purpose of water solubility (Scheme 1). Although, a number of sensors with their biological applications of copper are known but carbohydrate-based sensors for metal ions, including Cu2+ , are scarce in the literature [17–21]. In recent past few workers exploited the carbohydrate bearing chemosensors for metal ion recognition by involving glucosamine, chitosan, triazole and aza sugar derivatives [17]. Rao et al. explored some efficient chemosensors incorporating glucosamine/galactosyl-imine and -amine derivatives for detection of Fe3+ , Cu2+ and Hg2+ ions/amino acids [18]. Recently Duan et al. introduced chitosan over rhodamine and fluorescein for selective detection of Hg2+ and Fe3+ , respectively [17b,19]. Duan et al. also designed a Hg2+ selective probe based on conjugation of the glucosamine with quinolone [20] while Du et al. explored a napthaldehyde linked sugar moiety conjugated with rhodamine as a turn ‘on’ fluorescent sensor for Cu2+ [21]. Furthermore among sugar derivatives the uses of glucose/galactose moieties are not well explored as compared to other sugar derivatives. Duan et al. reported a fluorescent turn ‘on’ sensor (RG1) for Hg2+ by simply reacting glucose unit and Rhodamine 6G hydrazide [22]. Du et al. explored the same RG1 to detect Cu2+ by using different solvents for sensing studies [23]. Recently Zhang et al. reported three new fluorescent chemosensors bearing rhodamine B and sugar groups, viz., glucose, xylose and arabinose for selective detection of Cu2+ [24]. Thus we found only above two instances of incorporation of glucose moiety as such as a part of any chemosensor while no report was found in the literature for the use of galactose as such for the same purpose to the best of our knowledge. Herein, we utilized galactose as sugar unit for the first time over fluorescein to get a sugar–fluorescein conjugate FG as a highly selective and sensitive fluorescence ‘turn-on’ probe for Cu2+ ion with nanomolar sensitivity in aqueous medium at physiological pH. The FG is capable of showing both a chromo- and fluorogenic responses through opening of spirolactam ring of the FG in the presence of Cu2+ . Our considerations for synthesizing FG are as follows: (a) introducing galactose as sugar moiety not only improved the chelating ability, water compatibility, but also showed plausible

FG was synthesized via a simple condensation reaction, and characterized by 1 H NMR, 13 C NMR, IR and mass spectral studies (ESI; Figs. S1–S4). In a round bottom flask, Fluorescein hydrazide (1 mmol) and galactose (1 mmol) were suspended in 15 mL ethanol. The mixture was refluxed overnight. The reaction mixture was further evaporated to yield a sticky mass, which was washed several times with diethyl ether and finally recrystallized from methanol and dried under vacuum. Various spectroscopic data indicated that the galactose primarily condensed with fluorescein hydrazide to give corresponding Schiff base product (SB) which underwent rearrangement to yield the final product in the form of FG (Scheme 2). The 1 H NMR spectrum of FG showed a singlet broad peak at 6.089 ı ppm which was assigned as NH while no peak for aldimine CH N was observed. The same NH peak was not observed after deuteration of FG (ESI; Fig. S5) confirming that the FG incorporates cyclic form of galactose. 2.1.1. Important spectroscopic data for FG Yield = 68%; 1 H NMR (300 MHz, DMSO-d6 ), ı (ppm): 3.31–3.75, 4.22, 4.36–4.40, 4.47, 4.89, 6.08, 6.36–6.42, 6.57, 6.97, 7.47, 7.75, 9.81; 13 C NMR (75 MHz, DMSO-d6 ), ı (ppm): 60.56, 62.68, 64.63, 68.27–79.40, 82.53, 92.59, 97.50, 101.67, 102.36, 109.48, 109.93, 112.00, 122.35, 123.41–132.60, 151.51–152.40, 158.19, 165.47–166.81; IR (max , KBr, cm−1 ): 3391, 3265, 2938, 1711, 1695, 1611, 1509, 1467, 1450, 1381, 1323, 1231, 1185, 1106, 1067, 1044, 995, 956, 839, 754; ESI-MS: calculated for C26 H24 N2 O9 is 508.48; found [M+H] = 509.2. 3. Results and discussion 3.1. UV–visible studies The photo physical properties of FG were primarily investigated through UV–visible studies upon addition of the chloride salts of various metal ions in 70% aqueous HEPES buffered solution (H2 O:CH3 CN = 70:30, v/v, pH = 7.4). The absorption spectra of 50 ␮M aqueous solution of FG could exhibit absorption bands in UV-region only at 275 nm with appearance of a weak shoulder at 289 nm, which was ascribed to the closed form of FG (Fig. 1a). Upon concomitant additions of Cu2+ (0–5 equiv.) to the solution of FG, the absorbance was significantly increased in visible region with appearance of broad absorption bands at 632 nm and 396 nm with a weak shoulder at 500 nm (Fig. 1b and c). These results indicated towards Cu2+ driven spirolactam ring opening of FG on the similar line of previous reports [13]. An obvious colour change was observed from colourless to blue during the course of addition (Fig. 2). Moreover the colour (Fig. 2) and UV–visible spectral pattern of FG (ESI; Fig. S6) were also perturbed in the presence of Hg2+ on the similar line of Cu2+ but higher amount of Hg2+ were required to saturate the solution. On the other hand no significant absorption and colour changes occurred in the presence of a number of alkali or alkaline earth metal ions, viz., Na+ , K+ Mg2+ , Ca2+ , Ba2+ and a considerable number of d-block metal ions, such as Mn2+ , Fe3+ ,

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Scheme 2. Synthetic pathway for FG.

Co2+ , Ni2+ , Zn2+ , Cd2+ as well as Al3+ and Pb2+ from p-block element (Figs. 1d and 2). The above results indicated that FG can serve as a “naked-eye” Cu2+ /Hg2+ indicator in near aqueous media with Cu2+ a priority for

FG. Nevertheless no isosbestic point was observed which may be a consequence of hyperchromic shifting of absorption band of FG (275 nm) itself along with generation of new absorptions in visible (396 nm) to towards NIR region (632 nm) (Fig. 1c).

Fig. 1. (a) Absorption spectra of 50 ␮M solution of FG, (b) changes in absorption spectrum of FG upon addition of 5 equiv. of Cu2+ , (c) UV–visible titration profile of FG upon gradual additions of Cu2+ , and (d) bar graph showing absorption intensity of FG upon addition of 10 equiv. of various metal ions (M2+/3+ ).

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Fig. 2. Naked-eye colour responses of 50 ␮M solution of FG with various metal ions (10 equiv. each).

Fig. 3. Colour changes of FG with various metal ions under UV light (365 nm).

3.2. Fluorescence studies To investigate the interaction between FG and metal ions further, we performed fluorescence titrations of FG with Cu2+ and Hg2+ ions in 70% aqueous HEPES buffered solution (H2 O:CH3 CN = 70:30, v/v, pH = 7.4). The 1 ␮M solution of FG showed very weak fluorescence in visible region at around 515 nm. However, upon increasing additions of 0–15 equiv. of Cu2+ , the same solution of FG showed strong blue–green fluorescence (Fig. 3) with an approximately ∼12fold enhancement in the fluorescence intensity attributable to the delocalization in the xanthenes induced by its Cu2+ binding. The emission band was finally developed at 515 nm upon saturation of Cu2+ addition to FG (Fig. 4a). No obvious fluorescence and colour changes were observed in the presence of a number of alkali or alkaline earth metal ions, viz., Na+ , K+ , Mg2+ , Ca2+ , Ba2+ , Al3+ and a considerable number of d-block metal ions such as Mn2+ , Fe3+ , Co2+ , Ni2+ , Zn2+ , Cd2+ , Hg2+ as well as Al3+ and Pb2+ from p-block elements (Fig. 3). During UV–visible titration studies since Hg2+ also responded towards FG besides Cu2+ hence we checked further the possibility of selectivity towards any one out of Cu2+ and Hg2+ . Interestingly upon concomitant additions of Hg2+ to the 1 ␮M solution of FG no obvious changes were observed in the emission profile of FG even upon addition of its 50 equiv. (ESI; Fig. S7). These results suggested although it is not possible to discriminate Cu2+ or Hg2+ by FG through its absorption or naked-eye colour changes (Figs. 1 and 2) but the emission behaviour of the same responded selectively for Cu2+ only. No change in emission pattern of FG was observed even upon lapse of time for more than one hour after the addition of Hg2+ to FG. We recorded the fluorescent spectral pattern of FG in the presence of Hg2+ at the higher concentration of FG, i.e., at 10 ␮M. Interestingly Hg2+ was able to perturb the emission spectral pattern of FG at this concentration on the similar line of Cu2+ (Fig. 4b). The emission band was finally developed at 518 nm upon saturation of FG by the Hg2+ addition to it. The emission titration pattern of Hg2+ and FG was a bit broader than that of Cu2+ (Fig. 4b). These results demonstrated that although Hg2+ was also able to bind with FG but the binding ability of the same is much less than that of Cu2+ under similar conditions and is highly dependent upon the reaction time and the concentration of FG. From the Job’s plot (Fig. 5a), a maximum fluorescence change was observed when the molar fraction of the sensor [FG] versus [Cu2+ ] + [FG] was 0.5, indicating a 1:1 binding stoichiometry

between FG and Cu2+ (Fig. 5a). The binding stoichiometries were further confirmed through mass spectral studies. The mass spectra of FG + Cu2+ /Hg2+ (ESI, Figs. S8 and S9) showed a peak (M+H) at 570.1 (calculated for FG-2H + Cu2+ = 569.06) and 727.2 (calculated for FG-2H + Hg2+ + H2 O = 726.11), respectively, which correspond to formation of a 1:1 complex in both cases. The binding constant for FG with Cu2+ was calculated from the non-linear fitting of both UV–visible and fluorescence titration data (ESI; Figs. S10 and S11) in 1:1 binding equation [26] as (1.02 ± 0.08) × 105 M−1 and (2.51 ± 0.23) × 106 M−1 with satisfactory correlation coefficient values R = 0.99314 and 0.99241, respectively. The detection limits of FG towards Cu2+ /Hg2+ were calculated using both UV–visible and fluorescence titration data according to the IUPAC definition [27]. The corresponding values of detection limits for the FG with Cu2+ /Hg2+ have been given in Table 1. The detection limit (LOD) value for Cu2+ /Hg2+ determined through UV–visible titration data are of similar order with slight superiority of Hg2+ (1.91 × 10−7 M) over Cu2+ (1.84 × 10−7 M). However, the detection limit determined through fluorescence titration data yielded nanomolar (6.32 × 10−9 M) detection ability of FG towards Cu2+ (Fig. 5b) while the same was less effective towards Hg2+ (2.96 × 10−7 M). The calibration curves for the determination of detection limits were given in ESI; Fig. S12. Although UV–visible studies clearly indicated neck to neck detection ability of FG for Cu2+ and Hg2+ but the same for fluorescence titration data establishes the high sensitivity for Cu2+ instead of Hg2+ . This proves that our system is sensitive enough to monitor Cu2+ concentrations in aqueous samples. The selectivity of FG for Cu2+ over a wide range of potentially competing ions, such as a good number of alkali/alkaline-earth metals (Na+ , K+ , Mg2+ , Ca2+ , Ba2+ , Al3+ ) and a number of d-block metal ions, viz., Mn2+ , Fe3+ , Co2+ , Ni2+ , Zn2+ , Cd2+ , Hg2+ as well as Al3+ and Pb2+ from p-block element was investigated by fluorescence Table 1 Linear response of FG and value of detection limit (LOD) with Cu2+ and Hg2+ calculated through UV–visible and fluorescence titration data. S. No.

Metal ion

Detection limit (LOD) (M)

Linearity range

Method

1. 2. 3. 4.

Cu2+ Hg2+ Cu2+ Hg2+

1.84 × 10−7 1.91 × 10−7 6.32 × 10−9 2.96 × 10−7

2.50 × 10−5 3.25 × 10−4 2.00 × 10−7 2.00 × 10−5

UV–visible UV–visible Fluorescence Fluorescence

to 5.00 × 10−5 to 5.00 × 10−4 to 7.00 × 10−7 to 6.00 × 10−5

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Fig. 4. (a) Emission profile of 1 ␮M of FG upon concomitant additions of Cu2+ and (b) fluorescence titration spectra of 10 ␮M FG upon gradual additions of Hg2+ (ex = 470 nm).

Fig. 5. (a) Job’s plot of Cu2+ with FG showing 1:1 stoichiometry. (b) Calibration curve of FG with Cu2+ for determination of detection limit (from fluorescence titration data).

spectroscopy (Fig. 6). The emission intensities were recorded at 515 nm within 1 min after the addition of these metal ions (10 equiv. each) in the solution of FG (1 ␮M). As shown in Fig. 6, no significant spectral changes occurred in the presence of above

mentioned ions, while the addition of Cu2+ ions into the solution of FG produced a dramatic enhancement of the fluorescence intensity, revealing that Cu2+ could have specific effects on the fluorescence spectral pattern of FG, and the Cu2+ -specific responses were not disturbed by the competitive ions. 3.3. Binding studies

Fig. 6. Representative competition experiment of 1 ␮M aqueous solution of FG with various metal ions (5 equiv. each).

In order to understand the complexation behaviour of FG with Cu2+ and Hg2+ ions, we performed 1 H NMR experiments in DMSOd6 solution. As Cu2+ is a paramagnetic ion, and hence is less suitable for 1 H NMR experiments, we decided to investigate the complexation of FG with Hg2+ only (Fig. 7). Both of the phenolic-OH of FG were observed at 9.811 ı ppm. However, the 1 H NMR of the FG + Hg2+ complex showed resonance of only one broad upfiled shifted proton at 9.657 ı ppm. These results indicated towards metal ion driven spirolactam ring opening of FG and subsequently intramolecular shifting of a phenolic proton leading to in situ imidol formation and finally its deprotonation after its metal ion binding (Fig. 7). The yet another significant change in 1 H NMR spectrum of the complex was the absence of peak for NH, which was present in FG at 6.089 ı ppm indicating the involvement of NH in binding with Hg2+ upon its deprotonation. The other protons of fluorescein maintained its usual position

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Fig. 7.

1

H NMR spectra of FG and FG + Hg2+ complex in DMSO-d6 solution; Significant changes were marked with dashed circles.

galactose as binding unit. We believe that this approach makes FG as a very promising choice for Cu2+ detection in water as the same is able to detect Cu2+ rapidly through naked-eye with nanomolar sensitivity in fluorescence measurement. The present design principle of FG may help in the development of more efficient water and biocompatible optical chemosensors based upon sugar platforms. Acknowledgements Authors are thankful to CSIR, New Delhi for financial assistance [01(2709)/13/EMR-II]. UD acknowledges UGC-BSR for granting meritorious fellowship. A.K. acknowledges UGC, New Delhi for DSK Postdoctoral Fellowship [F.4-2/2006(BSR)/13-398/2011(BSR)]. 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.2014.02.031. References

Fig. 8. Proposed binding mode of FG with Cu2+ /Hg2+ .

without any obvious changes after complexation. The OH protons of galactose were not very clear in the complex, which may be a consequence of sufficient broadening and shortening of the signals. The CH protons of galactose were observed at their regular positions few of them were merged with solvent peaks. These results indicated a significant structural and conformational change of the FG ring as a consequence of its Hg2+ complexation and explicate that the FG acts as tridentate chelator. Finally on the basis of Job’s plot, mass spectral and 1 H NMR studies of FG with Cu2+ /Hg2+ we proposed the following tentative chemical structure of metal binding with FG (Fig. 8). 4. Conclusion In conclusion, we synthesized a near water-soluble colorimetric and fluorescent sensor FG having fluorescein as fluorophore while

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Biographies Uzra Diwan is a PhD student at Banaras Hindu University. She received her master’s in 2010 from Banaras Hindu University. She is pursuing her PhD degree in the field of optical receptors for ionic analytes under the supervision of Prof. K.K. Upadhyay. Ajit Kumar obtained his BSc and MSc from Ranchi University, Ranchi and received his PhD in 2010 from Banaras Hindu University. Presently he is working as postdoctoral fellow in Banaras Hindu University with Prof. K.K. Upadhyay. His research interests include exploration of some new moieties for development of efficient synthetic optical chemosensors. Virendra Kumar obtained his BSc and MSc from University of Allahabad, Allahabad, and presently working for PhD degree in Banaras Hindu University with Prof. K.K. Upadhyay. His research interests include optical sensors for ionic analytes of biological importance. K.K. Upadhyay received his PhD in inorganic chemistry from Banaras Hindu University in 1994. He is now working as a professor in department of chemistry, Banaras Hindu University. His current interest ranges from optical sensors for ionic analytes and self-assembly through supramolecular architectures. P.K. Roychowdhury received his PhD in organic chemistry from Banaras Hindu University. Currently he is serving Chembiotek Research International, Kolkata as a director of Analytical Division.