Article pubs.acs.org/Organometallics
Ferrocene and Triazole-Appended Rhodamine Based Multisignaling Sensors for Hg2+ and Their Application in Live Cell Imaging C. Arivazhagan, Rosmita Borthakur, and Sundargopal Ghosh* Department of Chemistry, Indian Institute of Technology, Madras, Chennai 600036, India S Supporting Information *
ABSTRACT: Two triazole-appended ferrocene−rhodamine conjugates, C47H45N7O3Fe (2) and C49H49N7O3Fe (3), have been synthesized, and their electrochemical, optical, and metal cation sensing properties have been explored in aqueous medium. The newly synthesized receptors are simple, easily synthesizable, and display very high “turn on” fluorescence response for Hg2+ as well as I− in an aqueous environment. Quantification of the absorption titration analysis shows that the receptors 2 and 3 can detect the presence of Hg2+ even at very low concentrations (∼3 ppb). The mode of metal coordination has been studied by DFT calculations. Furthermore, the receptors 2 and 3 are less toxic toward MCF-7 cells and could detect intracellular Hg2+ by fluorescent imaging studies.
■
binding ability of the probe can be switched on and off.12 It is well known that the incorporation of an electron-rich ferrocenyl group into the rhodamine system can enhance the coordination ability of the hydrazine nitrogen atom to Hg2+ ions.13,14 Based on these considerations, we designed a multichannel chemosensor combining a rhodamine derivative with a reversible redox-active ferrocenyl group. Further, to increase the complexation ability toward Hg2+, a triazole moiety was introduced into the receptors as a coordinating site.15 In continuation of our work to develop new and efficient redoxactive chemosensors for toxic-metal ions,16−19 a rhodamine− ferrocene conjugate with a triazole moiety has been designed as a new multichannel probe (Scheme 1) for detection of Hg2+. As expected, on addition of Hg2+, the probes showed significant switching-on fluorescence behavior and changes in their
INTRODUCTION Mercury, a toxic heavy metal, causes severe health problems such as immuno/geno/neurotoxic1,2 defects. Organic methyl mercury (CH3Hg+) formed from both elemental and ionic mercury by bacteria causes severe damage to the central nervous system, endocrine system, kidneys, skin, and DNA3−6 due to its accumulating in the human body through the food chain. Considering these environmental and health problems associated with Hg2+, it is a challenge to detect this toxic metal ion using new techniques where traditional techniques have failed.7 Thus, it is important to develop new techniques for its detection in vitro and in vivo. Several chemosensors have been developed for sensing Hg2+; however, due to their poor solubility in aqueous medium,1 these chemosensors could not be used for intracellular detection of Hg2+. In contrast to the advancement of individual optical-signaling sensors for Hg2+, there has been few reports regarding multisignaling probes.8 This encouraged us to develop a new multisignaling sensor for in vitro detection of Hg2+. An important practical challenge is to develop a fluorescent probe that shows enhanced fluorescence upon addition of Hg2+.1 Rhodamine-containing dyes are excellent fluorescent probes that have been widely used as an efficient dual-responsive sensor via chromogenic and fluorogenic signals.7,9 Several rhodamine derivatives have been successfully fabricated as OFF-ON-type fluorescent chemosensors due to the equilibrium between the spirolactam (nonfluorescence) and ring-opened amide (fluorescence) form.10 On the other hand, ferrocenebased receptors are suitable redox-active building blocks that can be easily functionalized and incorporated into various structures.11 Such systems can electrochemically sense neutral and charged molecules due to the change in the Fe/Fe+ redox couple, and by varying the applied electrochemical potential the © XXXX American Chemical Society
Scheme 1. Synthetic Scheme for Receptors 2 and 3
Received: September 16, 2014
A
DOI: 10.1021/om500948c Organometallics XXXX, XXX, XXX−XXX
Article
Organometallics
and 3, respectively, also support this assumption (see Supporting Information). To evaluate the binding interaction of receptors 2 and 3, the absorption response behavior of the receptors was investigated in the presence of various metal salts (Na+, Mg2+, K+, Ca2+, Cr2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Pb2+, Cd2+, Hg2+, and Tl+) (Figures 2, S4). Interestingly, upon addition of Hg2+, 2 and 3 showed new maximum absorption bands at 528 and 556 nm, respectively (Figure 2B and D), which can be attributed to the fully delocalized xanthene moiety of rhodamine. In this case the solutions of 2 and 3 exhibited an obvious and characteristic color change from colorless to red and pink, respectively (Figure S5). Thus, receptors 2 and 3 can be used for the “naked-eye” detection of Hg2+. Notably, when an excess of other metal cations was separately added to the solutions of 2 and 3, no significant color change or change in the absorption spectrum was observed (Figure 2). On sequential titration of 2, the high-energy (HE) absorption band at 304 nm decreases, while the low-energy (LE) band at 528 nm increases. Similarly, for 3 the band at 320 nm decreases with the simultaneous increase of the band at 556 nm. From these UV−vis spectral studies, it is clear that chemosensors 2 and 3 show highly selective binding affinity in the ground state for Hg2+. From the UV−vis titration profile, the binding constant values and the stoichiometric ratio of 2 and 3 with Hg2+ can be determined using the well-known Benesi−Hilderbrand (BH) equation.22,23 The linear nature of the double-reciprocal plots (Figure S6) indeed confirms 1:1 stoichiometry of the complexes, and the binding constant (K) values were found to be 5.07 × 104 M−1 and 4.25 × 104 M−1 for 2·Hg2+and 3· Hg2+, respectively. The formation of 1:1 (cation/receptor) stoichiometry of 2·Hg2+ and 3·Hg2+ was also confirmed by Job’s plot (Figure S7). Similar stoichiometry of the complexes was further confirmed by ESI-MS, where peaks at m/z 1013 and 1041 in the mass spectra correspond to the 1:1 complex of 2 and 3, respectively (Figures S8 and S9). The detection limit (DL) of 2 and 3 for the analysis of the Hg2+ ion is also an important parameter for practical purposes and can be determined from the fluorescence titration data using the protocol described elswhere.24 The measured DL was found to be ∼2.28 ppb and ∼3.40 ppb for receptors 2 and 3, respectively (Figures S10 and S11). These low detection limits fully meet the requirements in cellular applications. Fluorescence Spectroscopic Studies. The complexation ability of Hg2+ with the receptors was also investigated using fluorescence titration in CH3CN/HEPES buffer (2:8, v/v, pH 7.3, 10 μM). Both the receptors exhibited no obvious fluorescence in the range 500 to 700 nm in the absence of metal ions when excited at λex = 480 nm for 2 (Φf = 0.05) and λex = 495 nm for 3 (Φf = 0.02). This further supports the nonexistence of the fully conjugated xanthene form and the presence of the spirolactam form of the receptors in the absence of metal ions (Figure 3). Upon addition of Hg2+, receptors 2 and 3 induce significant switching ON of fluorescence response at 554 and 585 nm with a high fluorescence quantum yield Φf = 0.73 and 0.34 (Figure 3A and C), with a color change to yellow and purple, respectively (Figure S12). This switch ON response behavior of 2 and 3 on the absorption and emission spectra can be explained in terms of the spirolactam ring opening of the rhodamine moiety upon coordination with a metal ion. It was also evident from Figure 3 that the ring opening of 2 and 3
electrochemical properties. Furthermore, the chemosensors have been successfully used for monitoring Hg2+ as well as I− ions in living cells. In this article, we report the synthesis and characterization of receptors 2 and 3 along with their applications as multichannel sensors for Hg2+ ions in aqueous medium with a very low limit of detection, ∼3 ppb.
■
RESULTS AND DISCUSSION Mono(azidomethyl)ferrocene and 2-(prop-2-ynyloxy)benzaldehyde were synthesized as per the literature procedures.20a,b Compound 1 was synthesized by “click reaction” of mono(azidomethyl)ferrocene and 2-(prop-2-ynyloxy)benzaldehyde in the presence of copper(I) catalyst with 85% yield. Receptors 2 and 3 were synthesized by refluxing 1 with rhodamine 6G hydrazide and rhodamine B hydrazide, respectively, in 83−85% yield (Scheme 1). Receptors 2 and 3 are stable and have been characterized by usual spectroscopic techniques such as IR, 1H and 13C NMR spectroscopy, and ESI-MS (see Supporting Information). Further, the structure of compound 1 has been ascertained by X-ray diffraction analysis (Figure 1). The cation recognition properties of the receptors
Figure 1. X-ray structure of compound 1. Selected bond lengths (Å) and angles (deg): C10−C11 1.488(4), C11−N1 1.467(4), [triazole ring N1−C12 1.343(3), N2N3 1.313(4), N3−C13 1.346(3), N1− N2 1.327(3), C12−C13 1.352(4)], C13−C14 1.485(3), C14−O1 1.423(3), O1−C15 1.363(3); C10−C11−N1 113.0(2), C13−C14− O1 107.2(2), C14−O1−C15 117.80(18), [triazole ring N1−N2−N3 106.9(2), N2−N3−C13 109.0(2), N1−C12−C13 105.0(2)].
have been established by absorption, fluorescence, 1H NMR titration, and electrochemical studies. Further, receptors have been used as imaging probes for intracellular detection of Hg2+ and I− in human breast cancer cells (MCF-7).21 Solid-State X-ray Structure of 1. Compound 1 crystallizes in the monoclinic centrosymmetric C2/c space group (Figure 1). The two cyclopentadiene rings in compound 1 are arranged in an eclipsed form. As depicted in Figure 1, the bond distances of C−N in the triazole ring are in the normal range (average ∼1.344 Å). In the triazole ring the N2−N3 bond distance is slightly shorter than that of N1−N2; this is due to the nature of the double-bond character of the N2−N3 bond (Table S1). UV−Vis Absorption Studies. The UV−vis absorption studies have been performed using a CH3CN/HEPES buffer (2:8, v/v, pH 7.3, 10 μM) solution. The UV−vis spectra of 2 and 3 showed absorption maxima at 304 and 320 nm, respectively, due to an intramolecular π−π* charge transfer (CT) transition. No absorption peak was observed in the visible wavelength region above 400 nm, which indicates that both the receptors exist in a spirocyclic form in solution (Figure 2). The characteristic 13C NMR chemical shifts at δ = 65.75 and 66.03 ppm for the tertiary carbon of the spirolactam ring of 2 B
DOI: 10.1021/om500948c Organometallics XXXX, XXX, XXX−XXX
Article
Organometallics
Figure 2. UV−vis absorption spectra of receptors 2 and 3 (10 μM) observed upon addition of 3 equiv of various cations in CH3CN/HEPES buffer (2:8, v/v, pH 7.3, 10 μM) (A and C). Absorption titration spectra of 2 and 3 (10 μM) with Hg(ClO4)2 (0.0−1.0 equiv) in CH3CN/HEPES buffer (2:8, v/v, pH 7.3, 10 μM) (B and D).
immediately decreased upon addition of I− and S2− due to their strong affinity for the Hg2+ ion. For further investigation, three alternate cycles of titration of [2-Hg2+] and [3-Hg2+] were carried out by introducing I− into the system (Figure S15). This results in quenching of fluorescence due to the formation of a HgI2 complex and thus acts as an OFF switch. However, titration of the receptors with Hg2+ resulted in [2-Hg2+] and [3Hg2+] complex formation, which is accompanied by significant increase in the fluorescence intensities, thereby acting as an ON switch. The high degree of reversibility of the complexation/ decomplexation process of the receptors with Hg2+ can be achieved several times without a loss of sensitivity of the fluorescence intensity. The MALDI-MS of the solutions of [2Hg2+] and [3-Hg2+] containing I− revealed that the peaks at 812.408 and 840.637 correspond to free receptors 2 and 3, respectively (Figures S16 and S17). Electrochemical Studies. Ferrocene-containing chemosensors have been broadly studied as electrochemical sensors for metal cations.25,26 The metal cation-recognition properties of 2 and 3 in the presence of various metal ions have been investigated by cyclic voltammetry, differential pulse voltammetry (DPV), and linear sweep voltammetry (LSV) in CH3CN containing 0.1 M [(n-C4H9)4NClO4] (TBAP) as supporting electrolyte. As expected, receptors 2 and 3 displayed a reversible one-electron oxidation process at E1/2 = 0.57 V, due to the ferrocene/ferrocenium redox couple.
induced by metal−receptor coordination and the formation of the xanthene moiety is sensitive and selective toward Hg2+. Other cations such as Na+, Mg2+, K+, Ca2+, Cr2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Pb2+, Cd2+, and Tl+ exerted little or no effect on the emission spectrum (Figures 3A,C and S13). To further understand the sensing properties of 2 and 3 for Hg2+, a titration of the receptors was carried out with incremental addition of Hg2+. Upon addition of 1.0 equiv of Hg2+, the fluorescence intensity of 2 significantly increased (∼about 500-fold) (Figure 3B). This also confirms that receptor 2 exhibits a high sensitivity toward Hg2+. Similar results were observed for receptor 3 (Figure 3D). To check the practical ability of 2 and 3 as sensitive and selective fluorescent sensors, we carried out competitive experiments in the presence of Hg2+ ions mixed with alkali, alkaline, transition, and heavier transition metal ions. As illustrated in Figures 4 and 5, no significant variation in the fluorescence emission was observed. Thus, the receptors behave as selective chemosensors for Hg2+ in the presence of other competitive cations. To test the reversibility and reusability of 2 and 3 toward Hg2+, we carried out systematic absorption and fluorescence titration studies. Various anions (F−, Cl−, Br−, I−, CN−, CO3−, NO3−, NO2−, PO43−, SO42−, AcO−, and S2−) (Figures 6 and S14) were added to the solution containing the probe and Hg2+, and their absorption and fluorescence intensity changes were monitored after 5 min. Among all the anions, the enhanced emission due to Hg2+ coordination to the probe C
DOI: 10.1021/om500948c Organometallics XXXX, XXX, XXX−XXX
Article
Organometallics
Figure 3. Fluorescence spectra of receptors 2 and 3 (10 μM) observed upon addition of 3 equiv of various metal ions in CH3CN/HEPES buffer (2:8, v/v, pH 7.3, 10 μM), A (λex = 480 nm) and C (λex = 495 nm), respectively. Fluorescence titration spectra of 2 and 3 (10 μM) upon incremental addition of Hg(ClO4)2 in CH3CN/HEPES buffer (2:8, v/v, pH 7.3, 10 μM), B (λex = 480 nm) and D (λex = 495 nm), respectively.
No changes in CV and DPV of the receptors was observed upon addition of Na+, Mg2+, K+, Ca2+, Cr2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Pb2+, Cd2+, and Tl+ (Figure S18). However, as shown in Figures 7 and 8, a gradual decrease of the original peak upon stepwise addition of 1 equiv of Hg2+ induced a significant anodic shift of the Fe/Fe+ redox couple, while a new peak with increasing intensity was observed at 0.75 V (ΔE1/2 = 200 mV) for 2 and 0.80 V (ΔE1/2 = 250 mV) for 3 due to the formation of a complex species. The linear sweep voltammetry studies (Figure S19) further revealed similar results to those obtained from CV. 1 H NMR Titration Studies. In order to understand the binding mechanism, 1H NMR titration was performed by concomitant addition of Hg2+ to the CD3CN solution of 2. As illustrated in Figure 9, significant shifts in the 1H NMR spectrum were observed upon addition of Hg2+. After 5 min of addition of Hg2+ to 2, the peaks related to protons in the triazole ring and the methylene protons (Ha and Hb) were shifted downfield by ca. 0.35, 0.30, and 0.20 ppm, respectively. Such downfield shifts of the metal complex of 2 suggest that Hg2+ was chelated by nitrogen atoms of the triazole ring, imine, and the oxygen atoms, which are directly attached to the methylene proton and amide carbonyl group (Figure 9). To support the mode of binding, we have further performed IR measurements of 2 and 3 in the absence and presence of Hg2+ (Figures S20 and S21). The IR spectrum of 2 shows that the characteristic stretching frequency for the amide carbonyl (CO) of the rhodamine unit at 1710 cm−1 shifts to 1705
Figure 4. Results of the competition experiments between Hg(II) and selected metal ions. The free receptor 2 concentration was set at 10 μM, and the excitation was at 480 nm with a slit width of 5.0 nm. Solvent: CH3CN/HEPES buffer (2:8, v/v, pH 7.3, 10 μM).
Figure 5. Results of the competition experiments between Hg(II) and selected metal ions. The free receptor 3 concentration was set at 10 μM, and the excitation was at 495 nm with a slit width of 5.0 nm. Solvent: CH3CN/HEPES buffer (2:8, v/v, pH 7.3, 10 μM).
D
DOI: 10.1021/om500948c Organometallics XXXX, XXX, XXX−XXX
Article
Organometallics
Figure 6. Emission spectra of complexes 2-Hg2+ (left) and 3-Hg2+ (right) (10 μM) observed upon addition of 2 equiv of various anions in CH3CN/ HEPES buffer (2:8, v/v, pH 7.3, 0.1 mM).
Figure 7. Evolution of CV (left) and DPV(right) of 2 (c = 100 μM in CH3CN) upon addition of increasing amounts of Hg2+ up to 1 equiv using [(n-C4H9)4]4NClO4 as supporting electrolyte and scanned at 100 mV s−1.
Figure 8. Evolution of the CV (left) of 3 in the absence and presence of 1 equiv of Hg2+ and DPV (right) of 3 (c = 100 μM in CH3CN) upon addition of increasing amounts of Hg2+ up to 1 equiv using [(n-C4H9)4]4NClO4 as supporting electrolyte (scanned at 100 mV s−1).
cm−1 on addition of 3 equiv of the Hg2+ ion. Such shift is in accord with earlier reports.27 This shows that the spirolactam ring opens when Hg2+ coordinates to the receptors. DFT Studies. The mode of binding of Hg2+ with the receptors was also studied by DFT calculations (Figure S22). The stoichiometry of the complexes was found to be 1:1 on the
basis of absorption, emission, and ESI-MS studies, and these mononuclear complexes were modeled by DFT calculations. The geometry optimization for free receptors and their corresponding complexes was done by DFT calculations. For metal complexes, ground-state-optimized structures of free receptors were generated and Hg2+ was kept well in the core of E
DOI: 10.1021/om500948c Organometallics XXXX, XXX, XXX−XXX
Article
Organometallics
individual low-energy complexation occurs between Hg2+ and N2O2 atoms of the free receptors. Hence, the interaction of N2O2 with Hg2+ could change the HOMO−LUMO energy levels, and the electronic transitions should happen between the ferrocene-centered HOMOs and the π*-type LUMOs, located in the rhodamine unit. Furthermore, the DFT studies showed that the energy gap between the HOMO−LUMO of the complexes becomes smaller on complexation, which correlates with the observed red shift in the absorption spectra. To study practical applicability, the pH effect on absorption and emission response of receptors 2 and 3 was evaluated over a wide pH range of 1.0−13.0 (Figures S23 and S24). These experiments reveal that receptors 2 and 3 do not show any change in the absorption as well as emission spectra in the pH range of 5 to 13, suggesting the existence of the spirocyclic form. On the other hand, in acidic conditions (pH < 5) both the receptors show a pronounced effect in the absorption and emission spectra due to opening of the spirolactam ring. Thus, receptors 2 and 3 can be employed for the detection of Hg2+ in the near-neutral pH range (pH 7.3). The quick response (
