Synthesis and characterization of chemosensor: Investigation of cyanide sensing and study as live cell imaging

1 Introduction

Noteworthy researches have been conducted in the recent past, for the advancement of artificial receptors which can detect anions selectively due to their indispensable environmental and biological activities [1–13]. Among all the anions cyanide is one of the most nerve-racking anion due to its highly potent and quickly acting poisonous character in nature. Due to the strong affinity of cyanide to bind with the iron which is found in cytochrome-c-oxidase, it can therefore disrupt the transfer of electrons, which can result in hypoxia [14–21]. Cyanide has the potential to disrupt various biological activities in the human body, including the metabolic, cardiac, cardiorespiratory, endocrine, ocular and vital neurological systems [22]. On the other hand, cyanide has been widely used by humans in numerous chemical processes which include tanning, metallurgy, polymers, electro-plating and the withdrawal of silver and gold. Thus, the primary risk to the environment and the water contamination stems from the unintentional release of cyanide caused by all these technologies [23, 24]. As mentioned by World Health Organization that the concentration of cyanide with water must be lower than 1.9μM which is suitable for drinking [25]. Various analytical techniques which have been employed for detection CN^– such as potentiometry, chromatography and voltammetry are costly, rigorous, engage sophisticated instruments and painstaking procedures as well as involve well-trained professionals to handle the instruments. These are serious obstructions to the practical application of CN^– detection methods [26]. Consequently, the development of cyanide sensors that are affordable, easy to use, selective, responsive and inexpensive is of enormous demand now-a-days.

In this research work sensor A , (E)-(4-(2-((2-hydroxynaphthalene-1-yl)methylene)hydrazinyl)benzonitrile, has been employed for sensing of CN^– ion (as tetrabutylammonium salt) in double distilled water (DDW) and dimethyl sulfoxide (DMSO) (1 : 1 v/v) mixed solvent medium as well as imaging reagent to detect CN^– ion in SW480 cell line.

2 Experimental section

2.2 Synthesis of (E)-(4-(2-((2-hydroxynaphthalene-1-yl)methylene)hydrazinyl)benzonitrile (Sensor A)

To a solution of 4-cyanophenylhydrazine hydrochloride (0.394 g, 2.2 mmol) in ethanol (100 ml) solvent was added 2-hydroxy-1-naphthaldehyde, (0.4 g, 2.2 mmol) and 2–3 drops of acetic acid with continuous stirring. After addition of acetic acid, reaction mixture was allowed to reflux at 80°C for 8 hours. Reaction was monitored through TLC. After completion of reaction, reaction mixture was cooled to room-temperature. The precipitate was filtered and washed with ethanol. Finally, precipitate was dried in vacuum to obtain sensor A as creamy yellowish powder. Yield: 0.298 g (74%). IR (KBr-pellets, cm^–1): 3388 (O–H), 2218 (C≡N), 1608 (C = N). ESI-MS (m/z): 288 [M + H]⁺. ¹H-NMR (400 MHz, DMSO-d6, TMS) δ/ppm: 11.25 (s, 1 H), 11.09 (s, 1 H), 8.95 (s, 1 H), 8.6 (d, J = 8 Hz, 1 H), 7.88 (m, 2 H), 7.6 (d, J = 8 Hz, 2 H), 7.5 (t, J = 8 Hz, 1 H), 7.2 (d, J = 12 Hz, 1 H), 7.11 (m, 2 H). (Fig. S1–S3, Supplementary Information).

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Scheme 1

Synthesis of Sensor A .

As shown in scheme 1, the sensor A has been synthesized in excellent yield and the UV-Visible spectrum as shown in Fig. 1, represents a broad band at 385 nm in mixed solvent system of DDW/DMSO at ratio of 1 : 1. On steady adding up of CN^– ion, the UV-Visible band of A is red-shifted to 465 nm (longer wavelength). Alteration of absorbance at 465 nm with [CN^–] has been represented in inset of Fig. 1 which fits well in 1 : 1 stoichiometry using equation 1 shown in Fig. 2 to generate equilibrium constant of 3.7×10³ M⁻¹ [27, 28]. This equilibrium constant confirms the binding of CN^– ion with sensor A . Scheme 2 represents the binding of sensor A with CN^– ion, it has been determined from equilibrium constant which is derived from equation 1 for sensor A . The stoichiometry binding between the sensor A and CN^– ion is 1 : 1 which has been further determined through Job’s-plot method (Fig. Supplementary Information S4). The detection limit of chemosensor A with cyanide ion is found to be 39μM.

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Fig. 1

For Sensor A the spectral change in UV-Visible spectra has been observed on addition of CN^– ion in DDW/DMSO (1 : 1 v/v) on 298K. [ A ] = 1.6×10^–5 M, [CN^–] = (0 –5.7)×10^–4 M. Inset shows fitting (solid line) of the experimental data (change in absorbance for A at 465 nm (ΔA _465 nm) vs. [CN^–]).

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Fig. 2

Equations (1) and (2).

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Scheme 2

Proposed binding of sensor A with CN^– ion (Sensing Mechanism).

The binding between sensor A and CN^– ion is selective in nature has been established by adding DDW/DMSO (1 : 1 v/v) solution to a variety of different anions to fresh solution of the sensor A which has been displayed in Fig. S5 (Supplementary Information). Further, study of interaction has been performed between different ions like F^–, Cl^–, Br^– and I^– ions (25 equivalents) to the solution which contain CN^– ion (25 equivalent) and sensor A (Fig. S6 Supplementary Information). The study shows no alteration in the UV-Visible spectrum of solution further confirmed that the binding between CN^– ion and sensor A remains unaffected by interference of these ions. The fluorescence spectrum of sensor A shows a broad band of emission at 450 nm in DDW/DMSO (1 : 1 v/v) medium. As CN^– ion added to sensor A in DDW/DMSO (1 : 1 v/v) medium, new emission band is formed at 494 nm and the fluorescence spectrum intensity has been increased by 20 times (Fig. 3). The observed binding constant for sensor A with CN^– ion is 3.5×10³ M⁻¹. This binding constant was calculated with the help of Eq. 2 shown in Fig. 2 [27].

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Fig. 3

Changes in Fluorescence spectrum is observed for sensor A by the addition of CN^– in DDW/DMSO (1 : 1, v/v) at 298 K. [A] = 4.3×10^–6 M, [CN^–] = (0 –1.2)×10^–3 M,λ_ex=345 nm. Inset shows fitting (solid line) of the experimental data (fluorescence intensity ratio with and without addition of CN^– at 494 nm (I/I₀)_494 nm vs. [CN^–]).

The ¹H-NMR titration in DMSO-d₆ of sensor A with TBACN clearly shows the disappearance of the singlet signals of OH and NH on first addition of CN^– ion. Other proton signals of A are shifted up-field upon addition of CN^– ion. Sensor A has been studied for their colorimetric sensing ability in DDW/DMSO (1 : 1 v/v) solution by adding various anions. Colourless solution of A changed to yellow or light yellow on addition of 25 equivalents of CN^– ion in DDW/DMSO (1 : 1 v/v) solution, whereas on addition of 25 equivalents of F^–, Cl^–, Br^–, I^– and C₆H₅COO^– ions shows no change in colour (Fig. 4). Therefore, sensor A show selective sensing property for CN^– ion in DDW/DMSO (1 : 1 v/v) solution.

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Fig. 4

Colour changes observed for sensor A ([A] = 3.3 × 10⁻⁵ M), in DDW/DMSO (1 : 1 v/v) upon addition of 25 equivalents of different anions as TBA salts. From left to right: A , F^–, Cl^–, Br^–, I^–, CN^– and C₆H₅COO^–.

The biological application of chemosensor A has been estimated by treating the sensor to SW480 cells in absence and then in presence of CN^– ion. Fluorescence images of the live cells have been captured, as shown in Fig. 5. Chemosensor A display fine red coloured fluorescence with SW480 cells and its intensity got enhanced in presence of CN^– ion, therefore with the help of sensor A , CN^– ion can be detected intra-cellular in SW480 cells. Therefore, in the field of bio-analytical chemistry the sensor A can be exploited for quantitative as well as qualitative imaging of live cells affected by cyanide poisoning [29–34].

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Fig. 5

Fluorescence images of SW480 cell lines for sensor A (a) untreated cells, (b) bright-field image of cells treated with A (10μM) alone, (c) fluorescence image of cells treated with A (10μM), (d) bright-field image of cells treated with A (10μM) followed by CN^– (15μM) and (e) fluorescence image of cells treated with A (10μM) followed by CN^– (15μM).

Sensor A has been synthesized and characterized by FTIR, ¹H-NMR and ESI-MS spectroscopy. Sensor A binds with CN^– ion in DDW/DMSO (1 : 1 v/v) media selectively with significant change in UV-Visible and enhancement in fluorescence spectrum. OH and NH groups of sensor A bind CN^– ion through hydrogen bonding interaction which is supported by ¹H-NMR titration. DDW/DMSO (1 : 1 v/v) solution of sensor A displays outstanding change in color from colourless to light yellow on addition of CN^– ion. Sensor A act as imaging reagent for detection of the cellular uptake of CN^– ion in SW480 cell line.