Ajit Kumar Mahapatra*,
Kalipada Maiti,
Rajkishor Maji,
Saikat Kumar Manna,
Sanchita Mondal,
Syed Samim Ali and
Srimanta Manna
Department of Chemistry, Indian Institute of Engineering Science and Technology, Shibpur, Howrah-711103, West Bengal, India. E-mail: akmahapatra@rediffmail.com; Fax: +91 3326684564
First published on 26th February 2015
An indole conjugated thiophene–pyridyl (ITP) sensor for cyanide has been synthesized and characterized using NMR and mass spectroscopy. The selectivity of ITP has been explored in aqueous solution, and the resulting ratiometric fluorescence response toward CN−, among 11 different anions, was studied. The complexation of ITP–CN has been addressed using HRMS, 1H-NMR, and UV-vis spectroscopy. ITP displays substantial dual changes in both its ratiometric emission and absorption spectra, exclusively in the presence of CN− in aqueous solution. This is due to the nucleophilic attack of the indolium group of ITP by CN−, which induces a ratiometric fluorescence change and consequently a large emission shift. DFT/TDDFT calculations were performed in order to demonstrate the electronic properties of ITP and the ITP–CN adduct. The resultant ITP–CN adduct was used as a secondary sensing chemo-ensemble for the detection of cyanophilic metal ion-containing molecules by removing CN− from ITP–CN and regenerating ITP with switch-on red fluorescence. For the practical application of the sensor, test strips based on ITP were made up, which could act as suitable and proficient kits for CN− testing and cell studies.
Taking these considerations into account, several receptors have been proposed as optical sensors for cyanide ion detection.6 However, many of these sensors rely on a hydrogen-bonding motif in organic solvent and have generally displayed moderate selectivity over other anions.7
To overcome this problem, some reaction-based cyanide sensors have been designed and synthesized recently; these include oxazines,8 cationic borane derivatives,9 acridinium salts,10 and b-turn motifs.11 The fact is that many of them display either color changes or fluorescence changes, individually, but there are some limited examples where the receptors show simultaneous changes in both the absorption and emission spectra.7–13 Nevertheless, there are few chemical sensors14 that operate in water and show both the colorimetric and fluorescence changes upon their complexation with cyanide anions.15 Moreover, such sensors that undergo dual spectral changes show fluorescence quenching (On–Off) in their corresponding CN-adduct compound.11–14 So it is a challenge to the organic chemist to design and synthesize fluorescence chemosensors for cyanide that can show both color and fluorescence changes in an aqueous medium in a ratiometric manner.
Most of the reported cyanide sensors function as a result of fluorescence quenching or enhancement. As the change in fluorescence intensity is the only detected signal, factors such as instrumental efficiency, environmental conditions, and the probe concentration can interfere with the signal output. Ratiometric16 sensors have better utility than fluorescence-based chemosensors (On or Off) as they measure the fluorescence intensity at two different wavelengths, which increases the dynamic range and provides a built-in correction for environmental and concentration effects. In general, ratiometric sensors can function by the following two mechanisms: fluorescence resonance energy transfer (FRET)17 and intramolecular charge transfer (ICT).18 Of the two types, ICT based sensors are structurally much simpler and easier to make. Here, we report a new indole conjugated thiophene–pyridyl (ITP) probe, which shows ratiometric fluorescence changes with CN− by ICT blocking in aqueous solution.
The decision to use an indole moiety conjugated to a thiophene–pyridyl moiety as a chromogenic sensing molecule was mainly based on the fact that the conjugated indole skeleton could function as a color-reporting group. At the same time, conjugated indole based chemosensors show favorable photophysical properties, including an emission wavelength beyond 600 nm and a relatively large Stokes shift.19 So, the conjugation of indole with the thiophene–pyridyl moiety, as seen in Scheme 1, might modulate the intramolecular charge transfer (ICT) state and give rise to large dual color and fluorescence changes in the presence of CN−.
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Scheme 1 Scheme for the synthesis of ITP: (a) CH3I, CHCl3, r.t.; (b) 6-(2-thienyl)-2-pyridinecarboxaldehyde, EtOH, reflux. |
ITP shows intense red emission due to the extended π-conjugation and a strong ICT from the pyridyl moiety through the ethylenic group towards the indolyl moiety. Hence, it is expected that cyanide attack towards the indolium moiety not only interrupts the π-conjugation but also blocks the ICT process. This is why a hypsochromic shift occurs both in the absorbance and emission spectra of the ITP–CN adduct. To the best of our knowledge, only a few investigations on conjugated moiety-based ratiometric fluorescence versions of cyanide sensors have been reported before.20
The sensitivity of ITP toward different anions and its preferential selectivity toward CN− over the other anions was studied using fluorescence and absorption titrations. The ion recognition of ITP was studied by exciting the solutions (basic buffer at pH 9.3) at 411 nm and measuring their emission spectra from 450 to 800 nm. In order to ensure the nucleophilic addition of CN− to ITP, the fluorescence titrations were carried out in an aqueous basic buffer solution at pH 9.3, in 50 mM aqueous HEPES buffer taken with DMSO–H2O (5:
95, v/v) to give an effective buffer concentration of 10 mM. Among the 11 anions (n-Bu4N+ salts of F−, Cl−, Br−, I−, SO42−, HSO3−, AcO−, HS−, H2PO4−, and K+ salts of NO2− and CN−) only CN− caused a remarkable change in both visible color and fluorescence.
The titration of ITP with CN− resulted in a blue emission shift of 115 nm in the λem maximum of ITP. Titration by CN− also resulted in the gradual quenching of the fluorescence emission band at 619 nm, and a new emission band appeared at 504 nm as a function of the increasing CN− concentration in a ratiometric manner (Fig. 1). A clear isoemissive point at 556 nm also indicated the formation of the ITP–CN adduct.
We carried out a time-dependent cyanide adduct experiment on ITP (1.0 μM) in the presence of varying amounts of cyanide anion (0.5, 2.0, 5.0, and 10.0 μM) and observed a significant increase in the fluorescence intensity over the periods of 90, 65, 50, and 40 seconds, respectively (Fig. S14†). We also calculated rate constants for the abovementioned time-dependent measurements by plotting intensity versus time (Fig. S15†). The plot clearly demonstrates that the reaction follows pseudo first-order kinetics, and the rate constants for each concentration of CN− (0.5, 2.0, 5.0, and 10.0 μM) were found to be 0.026, 0.0258, 0.0261, and 0.026 s−1, respectively, which are almost the same irrespective of the cyanide concentration, as they should be. The minimum concentration of CN− that can be detected by ITP using fluorescence titration has been found to be 1.5 μM (Fig. S6†), signifying that the sensor can operate with concentrations well below the WHO cyanide standard in drinking water (1.9 μM).
In order to check whether ITP is sensitive to only CN− or even to the other ions, competitive anion fluorescence titrations were carried out in the same medium with 10 different anions, viz. F−, Cl−, Br−, I−, SO42−, HSO3−, AcO−, HS−, H2PO4−, and NO2−, and no significant fluorescence enhancement was found in the presence of these ions (Fig. 2a).
During the titrations, the ITP:
An− ratio was kept at 1
:
10 and the resultant solution was titrated against varying concentrations of CN− for each An−. It was seen that only HS− shows a little interference, while the other anions did not lead to any significant ratiometric fluorescence change, and the fluorescence emission spectra of ITP remained unaltered (Fig. 2b). Even the competitive titrations carried out by keeping the ITP
:
CN− ratio of 1
:
1 and varying the concentration of the other An− showed similar results.
In order to support the results obtained from the fluorescence studies, absorption titrations were carried out. The absorbance spectrum of ITP contained three characteristic bands at 246, 305, and 411 nm in buffer solution. While the absorbance values of the 305 and 411 nm bands decreased, that of the 246 nm band increased (Fig. 3a) on the gradual addition of the KCN solution up to 7 equiv. for saturation. Eventually, an obvious color change from dark brown to colorless was clearly observed, indicating that the ICT was turned off due to the nucleophilic attack by CN− at the indolyl cation of ITP. Thus the spectral changes and the isosbestic point observed at 260 nm clearly suggest the formation of a new species by a nucleophilic addition reaction between ITP and CN−, while there was no significant change in the absorption spectra of the other anions (Fig. S7†).
The CN− sensing property of ITP has been further supported by observing the fluorescent color change visually, in the presence of different anions under an incident light of 254 nm. Green fluorescence was found only in the case of CN−, while the other ions do not show such an emission (Fig. S13†). Furthermore, this has been carried out in the presence of other anions added to an initial solution possessing an ITP–CN adduct and no changes were found in the green fluorescence with any of the anions, suggesting that the CN− sensing by ITP can be monitored even in the presence of all the other more or less nucleophilic anions. The formation of a 1:
1 stoichiometric reaction product was confirmed using HRMS analysis, where a CH3CN solution of ITP with CN− showed a clear peak at m/z 394.1543 corresponding to [ITP + CN + Na]+ (Fig. S4†).
In addition, we also examined the 1H-NMR titration of ITP with CN− in d6-DMSO. After the addition of 2 equiv. of CN− (KCN) a significant proton shift occurred. However, the formation of ITP–CN led to an upfield shift of the olefinic proton, indicating that the electron withdrawing effect of the indole quaternary N atom decreased. The vinyl protons at δ 8.70 (Hi) and δ 8.45 (Hj) were upfield shifted to δ 8.28 and δ 8.23, respectively (Fig. 4). The N–CH3 protons of ITP were also magnetically deshielded due to the decreased electron density of the indole ring by the quaternization of the nitrogen atom and were shifted upfield after the addition of CN− (from δ 4.7 to δ 2.9).
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Fig. 4 1H-NMR chart (300 MHz, DMSO-d6, 0.5 mL) of ITP (10 mg) measured (a) without CN− and (b) with the addition of CN− (2.5 equiv.). |
To understand the absorption and fluorescence phenomena of ITP in the presence of CN−, density functional theory (DFT) calculations were performed using the Gaussian 03 suite of programs.21 Changes in fluorescence spectrum of ITP with the addition of CN− can be explained by time-dependent DFT (TDDFT) as well.
The calculated HOMO–LUMO energy gaps of ITP and ITP–CN are 2.30 and 3.82 eV, respectively (Table S1†). For ITP alone, there are two peaks in its absorption spectrum at 305 nm and 411 nm. These two bands arise due to the electronic transition of HOMO → LUMO+1 (3.93 eV/315 nm) and HOMO−1 → LUMO (3.06 eV/405 nm), respectively. The HOMO−1 → LUMO transition is the most fundamental transition, as it has a high transition percentage (64%) and a greater oscillator strength (0.71) (Table S1†). In the case of the ITP–CN adduct, the HOMO−1 → LUMO+1 transition is the most fundamental. The electron densities in the LUMO of ITP are distributed to the indolyl moiety through the thiophene–pyridyl (TP) moiety while, in the case of ITP–CN, the electron densities of LUMO, LUMO+1 and LUMO+2 only reside on the thiophene–pyridyl (TP) moiety.
There is a considerable difference in the energy minimization structure in ITP and the ITP–CN adduct, which can shed light on the changes in the absorption spectra and the corresponding changes in color. The energy minimization structure of ITP clearly shows the total planarity of the compound. Meanwhile, the energy minimization structure of ITP–CN shows that, after CN− attack, an indolyl group adopts a tilted geometry and becomes perpendicular to the pyridyl group (Fig. 5). This structural difference gives rise to the difference in π-conjugation between ITP and ITP–CN, and hence the ICT blocking. So, the shift in fluorescence towards the blue region in the ITP–CN adduct is mainly due to ICT blocking.
Owing to the well-known strong affinity of cyanide for Ag+, the weakly fluorescent cyanide-adduct [ITP–CN] has been studied for its properties in the secondary sensing of cyanophilic cations.
To our delight, the reversible process did happen and it can be understood to result from an aza-SN2′ displacement pathway triggered by Ag+. During the titration of ITP–CN with cyanophilic cations, only Ag+ caused a remarkable increase in the fluorescence emission band at 619 nm with the simultaneous quenching of the emission band observed at 504 nm (Fig. 6), which provides an alternative new approach for the undeveloped fluorescent sensing of silver ions. This is exactly the reverse of what happens when ITP is titrated with CN−, indicating the removal of CN− by Ag+ and thereby the release of free ITP. Inevitably, this is also the case with Cu2+, Au3+, and Au+, which also increased the emission intensity to a small extent and showed saturation at much higher equivalents. Thus, the ITP–CN complex acts as a secondary recognition ensemble for Ag+. The interaction of CN− with Ag+ and the consequent release of ITP were further supported using UV-visible absorption spectroscopy carried out with ITP–CN. The absorption spectrum obtained after the addition of Ag+ to ITP–CN is similar to that of free ITP, showing bands at 411 nm, 305 nm and 246 nm, suggesting the release of ITP from the adduct by Ag+ (Fig. S8†) (Scheme 2).
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Scheme 2 Schematic representation of the binding mode of cyanide with ITP and the regeneration of ITP from the ITP–CN adduct by Ag+. |
It can be observed with the naked eye that ITP shows characteristic color changes in the presence of cyanide in solution. This can be conveniently demonstrated using TLC plates which can in fact be further developed into handy test kits for the detection of cyanide ion. Test strips were prepared by immersing TLC plates into a water solution of ITP (c = 1.0 × 10−2 M) and then drying them in air. The test strips containing ITP were immersed in aqueous solutions with different cyanide concentrations to sense CN− and other anions. When the CN− ion concentration was increased, the color of the test strips changed from deep brown to colorless (Fig. 7). Additionally, potentially competitive ions did not influence the detection of CN− by the test strips. Similarly, fluorescent color changes from red to green occurred in aqueous solutions with different cyanide concentrations in the test papers, which indicates that they can provide a practical means of inspecting cyanide anion concentrations in the wilderness. In summary, the test strips could conveniently detect CN− in solutions. The above result suggests that this type of solid system may be used as a sensitive and practical “dip-in” naked eye cyanide sensor in the near future.
To demonstrate the practical application of the probe (ITP) to detect even a minute amount of CN−, we carried out experiments in living cells. In vitro studies established that the newly synthesized ITP probe can detect CN− with excellent selectivity even at concentrations as low as 50 μM. Hence, to assess the usefulness of ITP as a probe for the in vitro detection of CN− using confocal microscopy, RAW cells were used in the detection of CN− ions in live cells. We performed an MTT assay (Fig. S12†), which is based on monitoring the mitochondrial dehydrogenase activity of viable cells to study the cytotoxicity of the abovementioned compounds at the varying concentrations mentioned in the Experimental method section (†). Fig. 8 shows that the ITP probe did not exert any significant effect on cell viability; however the CN− ions had a dose-dependent adverse effect when cells were treated with varying concentrations of CN−. The ITP–CN complex also had a significant adverse effect on cell viability beyond 75 μM.
The exposure of HCT cells to the ITP–CN complex resulted in a decline in cell viability above a concentration of 20 μM. The effect was more pronounced in higher concentrations and showed an adverse cytotoxic effect in a dose-dependent manner.22 The viability of HCT cells was not influenced by the solvent (DMSO) (Fig. S12†), leading to the conclusion that the observed cytotoxic effect could be attributed to the ITP–CN complex. The results obtained in the in vitro cytotoxic assay suggested that, in order to pursue confocal imaging studies of the ITP–CN complex in live cells, it would be advisable to choose a working concentration of 10–20 μM for the probe compound. Hence, to assess the effectiveness of the compound ITP as a probe for the intracellular detection of CN− using confocal microscopy, RAW cells were treated with 20 μM CN− followed by a 10 μM probe solution to promote the formation of ITP–CN.
Confocal microscopy studies revealed a lack of fluorescence in the RAW cells when treated with CN− alone (Fig. 8b) and that red fluorescence developed when they were treated with ITP alone (Fig. S11†). Upon incubation with CN− followed by ITP, green fluorescence was observed inside the RAW cells, which indicates the formation of the ITP–CN complex, as observed earlier in the in vitro solution studies (Fig. 8c). Furthermore, an intense green fluorescence was noticeable in the perinuclear region of the RAW cells (Fig. 8d) and again red fluorescence reappeared upon treatment with an Ag+ solution (Fig. 8e). The confocal microscopy analysis strongly suggested that the ITP probe could readily cross the membrane barrier of the RAW cells, and rapidly sense intracellular CN− in very low concentrations. It is worth mentioning here that the bright field images of the treated cells did not reveal any gross morphological changes, which suggests that the RAW cells were viable. These findings open up an avenue for the future in vivo biomedical applications of this sensor.
In conclusion, we have synthesized an indole conjugated thiophene–pyridyl compound (ITP), which conveniently senses the CN− ion over other anions. Due to the high nucleophilicity of CN−, it selectively reacts with the indolium group to a greater extent than the other anions. ITP shows ratiometric fluorescence changes with the addition of CN− and has a large emission shift of about 115 nm. The high selectivity and fluorescence behavior of ITP was elucidated using DFT/TDDFT calculations. The detection limit of ITP is about 1.5 μM, which is lower than the maximum permissible level of CN− according to the WHO. In addition, test strips based on ITP were created, which also exhibited a good selectivity for CN− in water. The sensitivity of ITP is such that it can be used to monitor CN− in live RAW cells with ratiometric fluorescence imaging.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra17199c |
This journal is © The Royal Society of Chemistry 2015 |