Smart Probe for Multianalyte Signaling: Solvent Dependent Selective

Oct 26, 2015 - Available methods for fluorescence recognition of iodide mainly based on fluorescence quenching which is undesirable(25-29) due to the ...
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Smart probe for multi-analyte signaling: solvent dependent selective recognition of ISandip Nandi, and Debasis Das ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.5b00035 • Publication Date (Web): 26 Oct 2015 Downloaded from http://pubs.acs.org on October 28, 2015

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Smart probe for multi-analyte signaling: solvent dependent selective recognition of I− Sandip Nandi and Debasis Das* Department of Chemistry, The University of Burdwan, Burdwan, 713104, West Bengal, India. Correspondence: ; phone, +91-342- 2533913; fax, +91-342-2530452 KEYWORDS: azine based probes, Al3+, Zn2+, I−, real sample analyses ABSTRACT: An azine based smart probe is developed for naked eye and fluorescence recognition of I−. In both modes, the probe shows intense red color in presence of I− in THF. Moreover, the probe shows solvent dependent multiple signaling, viz. green emission with Al3+ in aqueous methanol and ratiometric yellow emission with Zn2+ in DMSO. The structure of the hydrazine precursor of the probe has been confirmed by single crystal X-ray structure.The probe can detect as low as 1.2×10−7 M I−, 6.8x10−8 M Al3+ and 5.7 x 10-8 M Zn2+ whereas respective association constants are 3.6× 104 M-1, 5.2 ×104 M−1 and 7.9 x 104 M−1. Common ions do not interfere. Routine Al3+ and Zn2+ imaging in human breast cancer cell, MCF7 under fluorescence microscope have also been performed. The developed method is used for analysis of real samples. Development of organic receptors for selective recognition of chemical and biological species is have great implications in chemistry, biology and environment.1, 2 Moreover, sensors whose fluorescence is sensitive to the solvent media and surrounding environment are specially interesting.3–6 The design of a molecular sensor depends on host–guest interaction promoted by hydrogen bonding, electrostatic force, metal–ligand coordination, hydrophobic and van der Waals interaction. A single molecule having different responses towards multiple analyte is cost effective and highly desirable for practical applications. However, the sensing of multiple analyte using a single probe is challenging, hence an active researcharea. As solvent may affect the physico−chemical nature of the receptor binding subunits, and hence the fluorescence output, the selectivity towards analyte is highly influenced by the solvatochromic nature of the receptor. This approach may be exploited to developsolvent dependent fluorescent sensor. Among bio-relevant anions, iodide (I−) is one of the essential micronutrients for normal human growth, particularly forproper functioning of thyroid gland.7 Na+/I− symporter (NIS) is an important plasma membrane glycoprotein that mediates active I− transport in the thyroid gland, the first step in thyroid hormone biogenesis.8 Deficiency or abundance of I− in the thyroid gland is a major health concern. According to the World Health Organization (WHO), iodine deficiency is the biggest global cause of mental retardation.9 On the other hand, Zn2+ plays diverse roles in medicinal, chemical, and biological events, most importantly a wellknown structural cofactor in metallo-proteins.10 Additionally, labile Zn2+ is responsible in signaling processes in brain, gene transcription and immunological functions.11−13 Its deficiency causes acrodermatitisenteropathica,14 while excess zinc may cause serious neurological disorders like Alzheimer’s and Parkinson’s diseases.15,16 Although, aluminum is extensively used in modern life, e.g. in food packaging, cookware, drinking water supplies, antiperspirants, deodorants, bleached flour, antacids and the manufacturing of cars and computers,17−19 its leaching from soil during acid rain increases free Al3+ in environment and surface water, detrimental to growing plants.20−22 Excess Al3+ exposure causes microcytic hypochromic anemia, bone softening, encephalopathy, myopathy and Alzheimer’s disease.23,24 Therefore, development of a single probe for trace level determination of these three ions, viz. I−, Zn2+ and Al3+ is very

challenging and demanding. Moreover, large ionic radius, low charge density and low hydrogen bonding ability make iodide, the most difficult anion to be sensed. Available methods for fluorescence recognition of iodide mainly based on fluorescence quenching which is undesirable25-29 due to possibility of false positive data by other possible fluorescence quenchers in the medium. Additionally, being heavy, iodide has intrinsic fluorescence quenching nature which is believed to be the principal reason for quenching. Few available fluorescence turn-on probes for iodide30 are “on−off−on” type that involves more steps, hence involve more error.31, 32 Although, individual sensors for detection of I−, Al3+ and Zn2+ are available, however, development of a single probe for detection of all three ions still remains unexplored. Herein, we report a new probe that allows solvent dependent colorimetric and fluorescence recognition of I−, Al3+ and Zn2+ at trace level. To rationalize and unfold the recognition process, two reference compounds have also been synthesized and studied.

EXPERIMENTAL Materials and methods High-purity HEPES, p-cresol, salicylaldehyde and benzaldehyde were purchased from Sigma–Aldrich (India). Zn(NO3)2·4H2O and Al(NO3)3·9H2O were purchased from Merck (India). The solvents used were of spectroscopic grade. 2, 6-Diformyl-4- methyl phenol was prepared by modification of a literature method.33 Metal salts were either as nitrate or chloride whereas anions were in the form of tetrabutylammonium salt. Other chemicals were of analytical reagent grade and used without further purification unless specified otherwise. Mili-Q Milipore 18.2 MΩcm−1water was used whenever required. A Shimadzu Multi Spec 2450 spectrophotometer was used for recording UV-Vis spectra. FTIR spectra were recorded on a Shimadzu FTIR (model IR Prestige 21 CE) spectrophotometer. Mass spectra were recorded using a QTOF 60 Micro YA 263 mass spectrometer in ES positive mode. The steady state emission and excitation spectra were recorded with a Hitachi F-4500 spectrofluorimeter. Elemental analyses were performed on a Perkin Elmer 2400 CHN analyzer. A Systronics digital pH meter (model 335) was used for pH measurement. Fluorescence microscope images were captured with an Olympus IX81 microscope and processed using image pro plus version 7.0 software.

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Synthesis Compound 3 The compound 3 was synthesized as shown in Scheme 1. To methanol solution of 2,6-diformyl-4-methylphenol (DFP, 0.41g, 2.5 mmol, 25 mL), excess hydrazine hydrate was added and stirred at room temperature for 6 h. After removing the solvent and excess hydrazine using rotary evaporator under reduced pressure, a light yellow solid was obtained. This product was dissolved in methanol and kept at room temperature for slow evaporation. After several days, rectangular yellow crystals suitable for single crystal X-ray diffraction were collected and used in the next step. Yield was 92%. The single crystal X-ray structure was presented in Figure S1 (ESI). Significant crystal and refinement parameters were summarized in Table S1 (ESI). QTOF–MS ES+ (Figure S2, ESI): [M + H]+ = 193.18. FTIR (cm−1) (Figure S3, ESI): ν(O–H, phenol) 3170, ν(N–H, 1° amine) 3321, Elemental analyses for C9H12N4O (192.10): calcd. (%) C, 56.24; H, 6.29 and N, 29.15; found (%), C, 56.43; H 6.23 and N 29.24.

Scheme 1

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well. After reaching 60%–70% confluence, the previous DMEM medium was replaced with serum free DMEM medium, supplemented with Al3+ (50 µm) or Zn2+ (30 µm) and incubated for 2 h to facilitate their cell uptake. Then the solution of probe 1 in respective solvent (20 µm) was added. The probe 1 treated cells were then incubated with Al3+ or Zn2+ for 15–30 min. The cells were washed thrice with PBS buffer to remove extracellular probe and metal salts and observed under the fluorescence microscope. The images were captured through an attached CCD camera equipped with BioWizard 4.2 software. The control experiment was performed similarly, devoid of any metal salts.

RESULTS AND DISCUSSION Probe 1 displayed a well-defined emission at 636 nm in aqueous methanol, DMSO and THF upon excitation at 390 nm (Figure S10, ESI). The pH of the medium was optimized for spectroscopic studies. For this purpose, probe 1 and Al3+ were mixed in different sets at different pH (pH 3.0 -11.0). Fig.S11 indicated a significant change of emission intensities in the pH ranging 4.0 to 11. However the optimum performance was observed in the pH ranging 4.0–8.0. Hence, pH 7.4 was maintained throughout Al3+ sensing studies for being closer to the physiological pH. Moreover, 0.1MHEPES buffered aqueous methanol (methanol/water, 1/1, v/v) was chosen as medium. Most common ions such as Na+, K+, Ca2+, Mg2+, Ni2+, Al3+, Co2+, Zn2+, Cd2+, Mn2+, Cu2+, Pb2+, Hg2+, Cr3+, Fe3+, AcO−, F−, Cl−, Br−, I−, CN−, ClO4−, H2PO4−, NO3−, SO42− and N3− did not show any significant spectral change (Figure 1), demonstrating the selectivityof probe 1 for Al3+.

Compounds 1 and 2 The probe 1 was synthesised by refluxing an equimolar mixture of salicylaldehyde and compound 3 in ethanol for 8 h (Scheme 1). A yellow solid was obtained after rotary evaporation of the solvent. Yield was 95%. Anal. calcd. (%): C, 68.99; H, 5.03 and N, 13.99; found: C, 69.07; H, 5.08 and N, 13.93. 1 H NMR (Figure S4, ESI) (500 MHz, DMSO-d6), δ (ppm): 12.14 (1H, s), 11.13 (2H, d, J = 15.5 Hz), 9.01 (4H, s), 7.72 (4H, m), 7.42 (2H, t, J = 6.0 Hz), 6.99 (4H, m, J = 15.0 Hz), 2.33 (3H, s).QTOF–MS ES+ (Figure S5, ESI): [M + Na]+ = 423.29. FTIR (cm−1) (Figure S6, ESI): ν(O–H, phenol) 3148, ν(CH=N) 1604. The compound 2 was synthesised by dissolving the compound 3 (2.5 mmol) in ethanol followed by addition of benzaldehyde (5 mmol) to the above solution and refluxing the mixture for 6 h to give a yellow compound. (Scheme 1) Yield was 91%. Anal. calcd (%): C, 74.98; H, 5.47 and N, 15.21; found: C, 75.09; H, 5.61 and N, 15.27. 1H NMR (Figure S7, ESI) (400 MHz, DMSO-d6), δ (ppm): 2.38 (3H, s), 7.53 (2H, m, J = 8.0 Hz), 7.57 (4H, d, J = 8 Hz), 7.90 (2H, m, J = 9.20 Hz), 7.95 (4H, t, J = 1.6 Hz), 8.75 (2H, s), 9.02 (2H, d, J = 2.0 Hz), QTOF–MS ES+ (Figure S8, ESI): [M + H]+ = 369.18. FTIR (cm−1) (Figure S9, ESI),ν(O–H, phenol), 3147; ν(CH=N), 1624. In vitro cell imaging MCF-7 cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C and 5% CO2. For in vitro imaging studies, the cells were seeded in six well tissue culture plates with a seeding density of 105 cells per

Figure 1 Changes in the (a) emission intensity of the probe 1 (20 µM) in presence of common ions, viz. Na+, K+, Ca2+, Mg2+, Ni2+, Al3+, Co2+, Zn2+, Cd2+, Mn2+, Cu2+, Pb2+, Hg2+, Cr3+, Fe3+, AcO-, F−, Cl−, Br−, I−, CN−, ClO4−, H2PO4−, NO3−, SO42− and N3− (50 µM); (b) corresponding UV light exposed colors. Medium: 0.1M HEPES buffered aqueous methanol (methanol/ water, 1/1, v/v, λex = 390 nm)

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Emission maxima of probe 1 (λex, 390 nm, λem, 636 nm) blue shifted by111 nm in presence of Al3+. Moreover, the emission intensity gradually increased upon addition of Al3+ (Figure 2).

To further strengthen the sensing mechanism, another receptor 2, lacking two hydroxyl groups was interacted with Al3+ and found negligible fluorescence change compared to probe 1 (Figure S18, ESI), suggesting the necessity of two –OH groups for efficient fluorescence enhancement. Binding of Al3+ with probe 1 was examined by 1H NMR titration in MeOD (Figure S19, ESI). Addition of one equiv. Al3+ to probe 1 did eliminate −OH protons completely whereas the imine protons were downfield shifted by 0.04 ppm and other protons remained unaltered. These indicated Al3+ coordination through two phenol oxygen and two imine nitrogen centers as proposed in Scheme 2.

Figure 2 Changes in the fluorescence spectra of probe 1 (20 µM) with increasing amount of Al3+ (0, 0.1, 0.5, 1, 2, 3, 5, 7, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50 µM) in the said medium (λex = 390 nm, λem = 525 nm).

The quantum yield of the probe 1 and its Al3+ adduct were 0.014 and 0.430. The plot of emission intensities of the probe1 as a function of added Al3+ was linear up to 15 µM (Figure S12, ESI), useful for determination of low level Al3+ .The emission profile of probe 1 and its Al3+ adduct remain unperturbed in presence of other competing ions (Figure S13, ESI). The binding affinity (Ka) of probe 1 for Al3+ was estimated as 5.2 ×104 M−1 from fluorescence titration exploiting the Benesi–Hildebrand equation34 (Figure S14, ESI) and Al3+ was detected down to 6.8x10−8 M, based on the 3σ/S method. The 1:1 binding interaction was observed between probe 1 and Al3+ from Job’s experiment (Figure S15, ESI) and ESI−MS spectrum (Figure S16, ESI). Fluorescence lifetime decay studies revealed that Al3+ enhanced the lifetime of probe 1 from 0.36 ns to 1.69 ns (Figure 3).

Figure 4 Changes in the absorption spectra of the probe 1 (20 µM) upon gradual addition of Al3+ (0, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40 and 50 µM) in the medium already mentioned.

Interestingly, changing solvent to DMSO did change the selectivity of probe 1 towards Zn2+. Very weak emission of probe 1 (λem = 636 nm, λex = 390 nm) changed significantly in presence of Zn2+ in a ratiometric manner whereby a new emission band at 538 nm increased gradually upon gradual increase of Zn2+ with the appearance of an iso-emissive point at 604 nm. Accordingly, the color of the solution turned yellow when exposed to UV light (Figure 5). The ratio of the emission intensities, F538 nm/ F636 nm varied linearly with added Zn2+ concentration (Figure S20, ESI). Moreover, fluorescence lifetime decay of probe 1 enhanced from 0.41 ns to 1.50 ns in presence of Zn2+ (Figure 6) and the fluorescence quantum yield increased from 0.018 (free probe 1) to 0.350 (1–Zn2+).

Figure 3 (A) Time resolved fluorescence decay of probe 1 in absence and presence of Al3+ in the medium already mentioned, (B) corresponding residual plot (λex = 390 nm, λem = 525 nm).

The absorbanceof the probe 1 was changed upon addition of Al3+ whereas other ions did not affect its absorbance significantly (Figure S17, ESI). The peak at 380 nm of free probe 1 disappeared upon addition of Al3+ with the appearance of a new metal to ligand charge transfer (MLCT) band ~420 nm, the absorbance of which increased with increasing Al3+ (Figure 4). Chelation with Al3+ inhibited both the CH=N isomerization and ESIPT processes, leading to fluorescence enhancement.

Scheme 2

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Figure 5 Changes in the (a) emission spectra of probe 1 (20 µM) in presence of common cations, viz.Na+, Ca2+, Mg2+, Ni2+, Co2+, Cd2+, Mn2+, Cu2+, Pb2+, Zn2+, Hg2+, Cr3+, Al3+, Fe3+ (30 µM) in DMSO; (b) UV light exposed colors of the probe 1 in presence of said cations. Inset: ratiometric change of the emission intensity of probe 1 (20 µM) with increasing Zn2+ concentration (0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 3, 5, 7, 10, 12, 15, 20, 25 and 30 µM (λex = 390 nm, λem = 538 nm).

Job's plot (Figure S21, ESI) indicated 1: 1 (mole ratio) binding of probe 1 with Zn2+, also corroborated from the mass spectrum of the [probe 1 - Zn2+] adduct (Figure S22, ESI). Association constant of the probe 1 for Zn2+ in DMSO was 7.9 x 104 M−1, determined using Benesi–Hildebrand equation34 (Figure S23, ESI). Common ions did not interfere in Zn2+ sensing (Figure S24, ESI). The probe 1 detected as low as 5.7 x 10-8 M Zn2+, nearly thousand fold lower than the WHO recommended tolerance level (76 µM) in drinking water.35

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solution (Figure S27, ESI), however instead of ratiometric sensing, normal fluorescence enhancement was observed (Figure S28, ESI). The plots of the emission intensities vs. added Zn2+ concentration was presented in Figure S29 (ESI), useful for unknown Zn2+ determination in aqueous DMSO media. The absorption spectrum of the probe 1 exhibited two characteristic peaks at 382 nm and 560 nm (Figure S30, ESI). Upon gradual addition of Zn2+, the absorption band at 560 and 382 nm were gradually decreased with the appearance of a new peak at 430 nm. Appearance of two iso-bestic points at 500 nm and 410 nm indicate the conversion of the free probe 1 into Zn2+ bound state through a common intermediate state (Figure 7). Thus, Zn2+ assisted selective fluorescence enhancement of probe 1 is attributed to the chelation enhanced fluorescence (CHEF) due to formation of rigid structure. Moreover, a prominent 98 nm blue shift indicated an inhibition of the ICT process in presence of Zn2+. The proposed CHEF mechanism was further substantiated with a control experiment using compound 2 that lacks two –OH groups. In presence of Zn2+, the emission spectrum of the compound 2 did not show any significant change (Figure S31, ESI), suggesting the necessity and involvement of the hydroxyl groups for Zn2+ sensing. For in depth understanding the binding mode of probe 1 with Zn2+, 1 H NMR titration was conducted in DMSO-d6 (Figure S32, ESI). Upon gradual addition of Zn2+ to probe 1, the imine protons were down-field shifted by 0.104 ppm along with disappearance of phenol proton of p-cresol moiety. Thus, probe 1 interacted with Zn2+ through imine nitrogen and deprotonated phenol oxygen centres.

Figure 7 Changes in the UV−vis. spectra of probe 1 (20 µM) upon gradual addition of Zn2+ (0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 3, 5, 7, 10, 12, 15, 20, 25 and 30 µM) in DMSO

Figure 6 (A) Time resolved fluorescence decay of probe 1 in the absence and presence of Zn2+, (B) corresponding residual plot (λex = 390 nm, λem = 538 nm).

The probe 1 could detect Zn2+ in aqueous DMSO media also. The effect of varying water content in the DMSO–buffer media on the emission intensity of 1-Zn2+ adduct showed that the emission intensity of the adduct decreased with increasing water percentage of the media (Figure S25, ESI). The effect of pH on the emission intensities of free probe 1 and its Zn2+ adduct indicated that the probe 1 could recognize Zn2+ over a wide pH range from 5.0 to 11.0 (Figure S26, ESI). Although the selectivity of probe 1 for Zn2+ remain unaltered in HEPES buffered (0.1 M, DMSO/water = 4/1, v/v, pH 7.5)

On the other hand, the probe 1 having four imine nitrogen and three –OH groups provided a very favorable platform for hydrogen bonding interactions with anions that might alter its electronic properties resulting a change of its emission properties. However, it is well known that hydrogen bonding interaction of anions with host molecule generally diminish in protic solvent. Hence, most of the anion recognition process was observed in non-aqueous and aprotic solvents. This was reflected in our present Al3+ and Zn2+ recognition studies where anions did not interfere (Figure S13 and Figure S24). Thus, we investigated the effect of anions on the emission properties of probe 1 both in DMSO and THF. Anions used as their tetrabutylammonium salts, TBAX, where X = F−, Cl−, Br−, I−, CN−, ClO4−, H2PO4−, AcO−, C2O42−, PO43−, NO3−, SO42− and N3−. Interestingly, in DMSO medium, three anions viz. I−, N3− and AcO− enhanced the emission intensity of probe

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1 to different extent while other anions did not show any effect (Figure S33, ESI). Surprisingly, change of solvent from DMSO to THF changed the selectivity to I− only which gave a turn-on fluorescence response associated with an instant color change from light yellow to red (Figure 8). Thus, the probe 1 function as a colorimetric and fluorescence sensor for I− in THF.

Figure 8 Changes in the (a) emission intensity of probe 1 (20 µM) in presence of various anions, F−, Cl−, Br−, I−, CN−, ClO4−, H2PO4−, NO3−, SO42−, H2AsO4−, HSO4-, AcO- and N3− (200 µM) in THF; (b) UV light exposed colors of probe 1 in presence of said anions (λex = 390 nm).

The fluorescence titration of probe 1 with I− enhanced the emission intensity at 636 nm (Figure 9) while the plot of emission intensities vs. I− concentration provided a calibration curve, useful for determination of unknown I− concentration (Figure S34, ESI). Job’s plot revealed a 1:1 stoichiometric interaction between probe 1 and I− (Figure S35, ESI). The association constant (K), determined following the described method was 3.6× 104 M-1 (Figure S36, ESI). The probe 1 could detect as low as 1.2×10−7 M I−. In presence of I−, fluorescence lifetime decay of probe 1 enhanced from 0.18 ns to 2.95 ns (Figure 10). Fluorescence quantum yield also increased from 0.011 (free 1) to 0.41 (1–I-) in THF. Selectivity for I− was further ascertained from the competitive experiment (Figure S37, ESI). It was found that the emission intensity of probe 1 in presence of 10 equivalents of I− remain unaffected upon addition of 10 equivalents of competing anions.

Figure 9 Changes in the fluorescence spectra of probe 1 (20 µM) with increasing concentration of I− (0, 0.5, 1, 3, 5, 10, 15, 20, 25, 50, 75, 100,125, 150 and 200 µM) in THF (λex= 390 nm, λem = 636 nm).

Figure S38 illustrated the change in the UV-Vis spectra of probe 1 in presence of common anions. The absorbance of

probe 1 (at 560 nm) increased 45 fold upon gradual addition of I− to a maximum of 10 equivalents (Figure S39, ESI). In presence of I−, the remarkable red color of probe 1 allowed naked eye detection of I−. The plot of absorbance (at 560 nm) as a function of I− concentration provided a linear region, useful for unknown I− determination (Figure S40, ESI).

Figure 10 (A) Time-resolved fluorescence decay of probe 1 in absence and presence of I−, (B) corresponding residual plot (λex = 390 nm, λem = 636 nm).

It was observed that upon addition of small amount of polar protic solvent like methanol or water, the red color disappeared. That was an indication of formation of hydrogen bond between probe 1 and I−. However, hydrogen bonding alone was not responsible for such a remarkable turn-on fluorescence as well as development of an intense red naked-eye color as other anions having hydrogen bonding ability including other halides would behave similarly. The preference for I− suggested that the pseudo-cavity formed by the probe 1 was more compatible to the size of I− than that of other anions having hydrogen bonding ability. Several reports guided us to conclude that size of the halide took predominant role to fit in the pseudo-cavity formed by the receptor binding sites,36-38 in contrary to their basicity, and hence H-bonding order, F−>Cl−> Br−> I− that would result a different observation. To further unfold the mode of host–guest interactions, 1H NMR titration was performed in DMSO-d6 (Figure S41, ESI). Appearance of the very downfield proton (-OH, d, 12.8 ppm) inprobe 1 was attributed to the intra−molecular H-bond between imine nitrogen and p-cresol-OH. Upon gradual addition of I− toprobe 1, the p-cresol-OH intensity decreased and eventually disappeared. No significant change of the chemical shift values of other protons was observed. Probably, acidic -OH proton of p-cresol exchanged rapidly with deuterium and hence, no change of its chemical shift value was observed. Replacement of the appended phenol part of the probe 1 by simple phenyl unit (compound 2) did not affect the selectivity towards I− in THF, only enhancement of emission intensity was relatively less (Figure S42, ESI). This indicates noninvolvement of –OH units of the appended phenol. Moreover, probe 1 was insensitive towards other species of iodine, viz. I2, IO4− and IO3− (Figure S43, ESI) The developed method had applied for analysis of Al3+, Zn2+ and I- in real samples. To evaluate the accuracy of the method, recovery studies of the said ions were performed at different concentration levels. Results were summarized in Table 1, Table 2 and Table 3. Sample preparation and analysis procedures were presented in ESI. Table 1 indicated excellent re-

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coveries of the analytes from both water and antacid samples, opening a new avenue for determination of Al3+ in pharmaceutical formulations and real water samples. The method was also employed for determination of Zn2+ in water and commercially available zinc gluconate samples (Table 2). The recovery of the method was ~98%, indicating the suitability of probe 1 for determination of Zn2+ in real samples. The percentage recovery of I- was evaluated in real urine sample and presented in Table 3. Table 1 Determination of Al3+ recovery in real samples. Sample Drinking water Antacid Suspension

Al 3+ added (µM) 4

Emission intensity (a. u.) 334.7

Al 3+ found (µM) 3.94

RSD (%)

Recovery (%)

1.8

98.5

3

529 684

7.28 9.97

2.3 2.1

97

Table 2 Determination of Zn2+ recovery in real samples. Zn2+ added (µM)

Emission intensity (a. u.)

Zn2+ found (µM)

RSD (%)

Recovery (%)

Drinking water

5

384

4.89

1.6

97.8

Zinc gluconate-oral suspension

-

395

5.29

1.9

-

3

493

8.11

2.1

98

Sample

Table 3 Determination of I- recovery in water and urine. Sample

Drinking water Urine





I added (µM)

Emission intensity (a. u.)

I found (µM)

RSD (%)

Recovery (%)

5 7

459 530

4.57 6.21

2.7 3.1

91.4 88.7

5

362 496

1.21 5.92

3.7 3.4

95.4

3+

2+

Probe 1 was applied to image Al and Zn in the human breast cancer cell, MCF7 following the literature procedure39. Cells remain non-fluorescent when treated with Al3+, Zn2+ and probe 1 separately, however, the cells containing Al3+ and Zn2+ emitted green and greenish-yellow light respectively after addition of the probe 1 (Figure11).

probe 1 (20 µM) + Al3+ (50 µM) and (f) cells + probe 1 (20 µM) + Zn2+ (30 µM).

CONCLUSION Thus, an azine derivative has been established as a solvent dependent multi-analyte sensor with smart recognition of I− both by colorimetric and fluorescence method. The probe generates intense red color with I− in THF, green emission in aqueous methanol with Al3+ and yellow emission with Zn2+ in DMSO. All analytical parameters and routine cell imaging studies have also been performed. Finally the probe has been used successfully for determination of the analytes in real samples.

ASSOCIATED CONTENT Supporting Information Contains 1HNMR and MS spectra of the probe and its adducts with analyte. It also contains single crystal X-ray structure of the precursor hydrazone derivative, associated crystal parameters, bond lengths and angles etc. Experimental details of different binding constants and LOD plots, interference studies etc. have also been presented here. This material is available free of charge via internet at http://pubs.acs.org. Notes The CCDC number of compound 3 is 1029420. The authors declare no competing financial interest.

ACKNOWLEDGMENT S. Nandi is grateful to UGC, New Delhi for fellowship. We are grateful to DST (Gov. of WB) for funding. We thank Dr A Banerjee, Department of Chemistry & Biochemistry, University of Texas at Austin, USA for routine cell imaging studies.

REFERENCES (1).

(2).

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(7).

Figure 11 Fluorescence microscope images of the MCF7 cells after incubation for 2 h (a) free cells, (b) cells + probe 1 (20 µM), (c) cells + Al3+ (50 µM), (d) cells + Zn2+ (30 µM), (e) cells +

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