Green synthesis of a benzothiazole based ‘turn-on’ type fluorimetric probe and its use for the selective detection of thiophenols in environmental samples and living cells

Abstract

We have developed an efficient turn-on type fluorescent chemodosimeter for the detection of aromatic thiols in aqueous media. The probe was successfully synthesized by condensation of 2-aminothiophenol with salicylaldehyde followed by S_NAr with 2,4-dinitrochlorobenzene in one-pot in a micellar medium. The function of the probe is attributed to the thiol driven cleavage of the dinitrophenyl ether linkage of probe 1 to release 2-(2-hydroxyphenyl)benzothiazole (2) which shows strong greenish fluorescence by excited-state intramolecular proton transfer (ESIPT). The probe is highly selective for aromatic thiols in the presence of several aliphatic thiols, including biologically important thiol-containing amino acids. The utility of the probe was successfully demonstrated in the detection of thiophenols in real samples and in living cells. A hazardless synthetic procedure, cost effective single-step synthesis, high selectivity and sensitivity, fast signal transduction and low limit of detection (3.3 ppb) are some of the key merits of this analytical tool.

---

1. Introduction

Thiophenols, although having a broad synthetic utility as insecticides, pesticides, in pharmaceuticals and in amber dyes,¹ belong to a class of highly toxic and pollutant materials.² In fact, thiophenols are much more toxic than their aliphatic analogues, e.g., for fish, thiophenols have a median lethal dose (LC₅₀) of 0.01 to 0.4 mM.³ Due to their high toxicity and ease of entrance into the human body by inhalation and skin absorption, thiophenols exacerbate grave systemic and central nervous injuries like burning sensation in the throat, eyes and nose, headache, dizziness, coughing, wheezing, laryngitis, shortness of breath, muscular weakness, paralysis, coma and even death.⁴ Thus, development of efficient techniques for the selective and sensitive detection of these toxic compounds in the environment is highly desirable.

The field of sensing and imaging agents is growing fast, largely driven by the development of optical techniques. In particular, the fluorimetric techniques have gained immense attention because of their operational simplicity, cost-effectiveness, high sensitivity and selectivity, fast signal transduction etc.⁵ Amongst available techniques for fluorimetric detection of thiols,^6–9 a large number of probes are designed for selective detection of biological thiols.^7,8 However, fluorimetric probes for selective detection of aromatic thiols are not many^8,9 as compared to biothiols because of their inefficiency to discriminate between aromatic and aliphatic thiols.^8a–e This imposes restriction to monitor only aromatic thiols in environmental and biological samples. In addition, some of these methods are not passably sensitive or selective, or involve relatively long synthetic route and thus, not cost effective. Some other available methods for the detection of aromatic thiols include HPLC technique,¹⁰ UV-detection and GC-MS methods,¹¹ optical spectroscopy using gold nano-particles as the probe,¹² chemically modified barium titanate beads using surface-enhanced Raman scattering (SERM),¹³ etc. However, these techniques suffer from expensive instrumentation and requirement of highly trained technicians.

Due to present environmental concerns use of greener solvents, nontoxic reagents and environment friendly conditions for any synthetic process has become imperative.¹⁴ Surprisingly, these issues are mostly ignored while the synthesis of molecular sensors. Although significant efforts have been made towards preparation of water soluble probes¹⁵ in order to reduce or eliminate the use of toxic organic solvents, there are hardly any attempts to maintain a “green” protocol during the synthesis of the probe molecule. In fact, the amount of organic solvents used during synthesis is often significantly higher than the amount required for analytical studies. In this work, we intended to introduce the concept of green chemical approaches in synthesizing the molecular probes. Thus, as a part of our continued efforts on the development of fluorescent chemosensors for environmentally and/or biologically toxic analytes,¹⁶ we report, herein, an environment friendly synthesis of a molecular probe which is highly selective and sensitive to aromatic thiols.

A large number of fluorescent probes have been constructed by exploiting the high nucleophilicity of the thiol group.^6–9 In many of these cases strongly electron-withdrawing 2,4-dinitrobenzenesulfonyl (DNBS) group has been used as the fluorescence quencher by getting attached with the fluorophore unit as sulphonate or sulphonamide.⁸ This strategy is effective in building chemosensors for selective detection of thiophenols, however, aliphatic thiols are often capable of detaching this moiety partially^8e or fully,^8a–d from the fluorophore at the prevalent condition because of the high lability of DNBS in the presence of –SH group making discrimination of thiophenols from other thiols difficult. We envisioned, use of 2,4-dinitrophenyl (DNP) group with reduced reactivity will be more effective in obtaining higher selectivity towards thiophenols and the corresponding probe molecule will be unperturbed by the presence of aliphatic thiols. The probe was constituted by protection of the free hydroxyl group of 2-(2-hydroxyphenyl)benzothiazole with DNP. We assumed, the aromatic thiols will remain partly in thiolate form at physiological pH and behave as much stronger nucleophile than aliphatic thiols. It would selectively cleave the ether linkage of the probe with ease releasing fluorescent 2-(2-hydroxyphenyl)benzothiazole, which will start emitting light by excited-state intramolecular proton transfer mechanism (ESIPT).¹⁷ The availability of the phenolic proton is essential for ESIPT to operate, which is unavailable in probe 1 due to formation of dinitrophenyl ether and the probe becomes nonfluorescent. When the probe comes in contact with thiophenols under the appropriate conditions, spontaneous trans-etherification occurs resulting in release of fluorescent 2-(2-hydroxyphenyl)benzothiazole (2). Probe 1 was obtained by mixing 2-aminothiophenol, benzaldehyde and 2,4-dinitrochlorobenzene in a single-pot in micellar medium and its sensing property was determined.

2. Experimental section

2.1 Chemicals and materials

All the reagents were purchased from commercial suppliers and used without further purification. AR grade solvents were used throughout the experiments. Doubly distilled water was used for the fluorimetric studies. Thiophenol, 2-aminothiophenol, 4-aminothiophenol were purchased from Spectrochem Pvt. Ltd. (India), SD Fine chemicals (India), Sigma Aldrich (India), respectively.

2.2 Instruments

NMR spectra were recorded on Bruker AV400 NMR spectrometer and mass spectra were obtained from Bruker micro TOF-Q II 10330 HRLCMS (ESI⁺). Fluorescence spectra were taken on a JASCO FP-6300 spectrofluorimeter with the slit width of 5 nm for both excitation and emission. Absorption spectra were recorded on a JASCO V570 UV/Vis/NIR spectrophotometer at room temperature. IR spectra were recorded on IR Affinity-1 FTIR spectrophotometer, Shimadzu. The reactions were monitored by thin layer chromatography (TLC) carried out on 0.25 mm silica gel on aluminium plates (60F-254) using UV light (254 or 365 nm) and could be tracked by naked eye.

2.3 Synthetic procedure

2.3.1 Synthesis of probe 1. In a 25 mL of round-bottomed flask was taken o-aminothiophenol (76 μL, 0.71 mmol) and salicylaldehyde (76 μL, 0.71 mmol) in 1 mL of water followed by addition of CTAB (26 mg, 0.071 mmol). The reaction mixture was stirred at room temperature for 1 h to give 2-(2-hydroxyphenyl)benzothiazole (2). The completion of the reaction was monitored by TLC. To the same reaction mixture 4 mL of water and K₂CO₃ (490 mg, 3.55 mmol) were added followed by addition of 2,4-dinitrochlorobenzene (142 mg, 0.71 mmol). The reaction mixture was sonicated for 5 min and stirred at room temperature for another 6 h. With the progress of the reaction solid product started separating out. After completion of the reaction, excess water (10 mL) was added and the residue was filtered, washed with water, and air dried. The crude product thus obtained was recrystallized from rectified spirit to afford probe 1 (180 mg, 64%). ¹H NMR (400 MHz, CDCl₃): δ (ppm) 6.93 (1H, d, J = 7.0 Hz), 7.23–7.27 (1H, m, merged with solvent peak), 7.36–7.39 (1H, m), 7.45–7.53 (2H, m), 7.56–7.59 (1H, m), 7.83 (1H, d, J = 6.2 Hz), 8.00 (1H, d, J = 6.2 Hz), 8.24 (1H, dd, J = 6.0, 1.2 Hz), 8.48 (1H, dd, J = 6.0, 1.2 Hz), 8.90 (1H, d, J = 2.0 Hz); ¹³C NMR (100 MHz): δ (ppm) 117.82, 121.49, 122.09, 122.21, 123.45, 125.70, 126.53, 126.58, 127.41, 128.94, 131.29, 132.48, 135.49, 139.43, 141.79, 150.70, 152.67, 155.63, 160.86; IR: 3098, 2914, 2855, 1610, 1536, 1342, 1251, 1145, 1099, 1067, 962 cm⁻¹; ESI-MS: m/z 394 [M + H]⁺; HRMS (ESI): m/z calcd for C₁₉H₁₁N₃O₅S [M + H]⁺ 394.0453, found 394.0452.

2.4 General procedure for the sensing studies

A stock solution of probe 1 (1 mM) was made in DMF. The fluorescence and absorbance studies were done by adding thiophenol to probe 1 in 45% DMF–PBS buffer solution (pH = 7.2) and incubating at room temperature for 15 min. To check the selectivity of the probe, the same procedure was followed with different thiol containing amino acids, 2-mercaptoethanol and other thiophenol derivatives. For equivalence study, the concentration of thiophenol was changed from 0–3.0 equiv. and fluorescence spectra were recorded.

2.5 Cytotoxicity

Cytotoxicity test of probe 1 was performed using MTT assay. HeLa cell lines were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum at 37 °C under humid condition with 5% CO₂. Cells were seeded in a 24 well plate at a density of 5 × 10⁴ cells per well. After 24 h of incubation, the cells were supplemented with probe 1 at different concentrations (10 μM, 50 μM and 100 μM; these solutions were prepared by adding 2 μL, 10 μL and 20 μL from 10 mM stock solutions of probe in 5% DMF–water). The control cells were treated with 0.1% of DMF. After incubation for 24 h, 60 μL of MTT (5 mg mL⁻¹ in phosphate buffer saline) was added per well and incubated for 4 h. After incubation, media was removed from the wells and 0.5 mL of DMSO was added to each well. Absorbance was measured at 570 nm and cell viability was expressed as relative absorbance (%) of the sample vs. control cells.

2.6 Fluorescence imaging of living cells

HeLa cells were seeded in a 6 well plate in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum for 24 h. HeLa cells were then incubated with thiophenol (1 μM) in the culture medium for 30 min at 37 °C. After washing with PBS buffer for three times to remove the remaining thiophenol, the cells were further incubated with probe 1 (1 μM) for 30 min at 37 °C. After incubation, the cells were again washed with PBS buffer for three times and the fluorescence images were obtained using Nikon eclipse TS100 microscope fitted with fluorescence attachment and using a FITC filter.

2.7 Real sample analysis

2.7.1 Detection of thiophenol in water samples. The presence of thiophenol was detected by spiking known amount of thiophenol in tap water. The water sample was filtered through microfiltration membrane. The pH of water sample was adjusted to 7.2 using PBS. Next, 1.5 mL of water sample was spiked with two different concentrations of thiophenol (1.5 μM and 3.0 μM) in DMF. These samples were further treated with probe 1 (30 μL, 1 mM) and diluted to 3 mL using DMF and water as per requirement. The solutions were incubated at room temperature for 15 min and the fluorescence spectra were measured.

2.7.2 Thiophenol vapor detection by paper strips and TLC plates. A paper strip of probe 1 was prepared by dipping a rectangular shape Whatmann filter paper in the probe solution (1 mM) in DMF for few seconds and then it was placed in chemical storage cabinet where the uncapped bottle of thiophenol was stored and allowed to get exposed to its vapor for 15 min. As a control experiment another paper strip was dipped in the same solution and kept in the storage cabinet without exposure of thiophenol for 15 min. The strip in contact with thiophenol showed strong fluorescence after 15 min of exposure. In a similar manner, probe 1 modified TLC plate (0.25 mm silica gel on aluminium plate) was prepared by spotting one drop of probe solution (1 mM) in DMF, allowed to soak properly, and then placed in the chemical storage cabinet for 15 min where an uncapped bottle of thiophenol was preplaced. The spotted area in TLC plate showed strong fluorescence. Like paper strips similar control experiment was conducted for TLC plates.

2.7.3 Detection of thiophenol in soil samples. To detect aromatic thiols in soil samples it was contaminated with measured amount of thiophenol from 1 mM stock solution in ethanol. In 3 mL DMF–PBS (pH = 7.2) containing probe 1 (1 mM) was added soil sample (90 mg) pre-spiked with thiophenol (1.2 μM) in ethanol. This sample was incubated at room temperature for 15 min, after which the solution was filtered for fluorescence measurement.

3. Results and discussions

3.1 Synthesis and spectral characterization of probe 1

Probe 1 was synthesized by condensation of 2-aminothiophenol with salicylaldehyde followed by aromatic nucleophilic substitution (S_NAr) with 2,4-dinitrochlorobenzene in one-pot in a micellar medium with 64% overall yield (Scheme 1). The formation of probe 1 was well-supported by ¹H NMR, ¹³C NMR, and HR-MS. As expected, the characteristic peak of aromatic proton flanked by two nitro groups appeared at the most downfield position (at δ 8.90 ppm) in ¹H NMR as a doublet with coupling constant (J) 2.0 Hz, which is attributed to meta-coupling. In ¹³C NMR, separate peaks appeared for each of the 19 carbons in the expected region indicating attachment of 2,4-dinitrophenyl unit to 2-(2-hydroxyphenyl)benzothiazole part. The molecular ion peak ([M + H]⁺) appeared as the base peak at m/z 394 firmly indicating formation of probe 1. In addition, HR-MS data for [M + H]⁺ of probe 1 closely matched with the expected value. In a separate reaction, 2-(2-hydroxyphenyl)benzothiazole (2) was isolated from the micellar medium in high yield (88%) and characterized by ¹H NMR, ¹³C NMR and HR-MS. Probe 1 was used for the detection of aromatic thiols.

Scheme 1 Synthesis of probe 1.

3.2 Effect of solvent and pH

To establish proper condition for thiolysis of dinitrophenyl moiety a thorough screening of solvent system was done as well as the effect of pH was studied. In this direction, experiments were performed varying different solvent systems to choose the best reaction medium for this aromatic nucleophilic substitution reaction. The reactions were carried out by adding thiophenol in a solution of probe 1 of a given solvent system in 1 : 1 molar ratio under neutral condition at room temperature. We used different proportions of organic solvents in water. Among DMF, THF and ethanol it was found that 45% DMF–water is most suitable for the system to smoothly undergo S_NAr reaction. Therefore, all further experiments were carried out in this solvent system. To check the effect of pH on probe 1, the reaction was done at various pH (pH 1–9). In a separate study, several solutions of probe 1 at different pH were prepared in 45% DMF–water and thiophenol was added to it, and fluorescence output of the resulting solution was measured after 15 min. Fig. 1 shows the fluorescence responses of probe 1 without and with thiophenol as a function of pH. The experiment revealed that the probe is ineffective in the detection of thiophenol at acidic pH (pH = 1–6) as expressed by very low fluorescence response from the solution. However, the same reaction when carried out under neutral condition using 45% DMF–PBS buffer (pH 7.2), to our delight, significant increase in fluorescence intensity was noticed. Under the prevailing condition the cleavage of the ether bond resulted in formation of fluorescent compound 2. On the other hand, under basic condition (pH ≥ 8), the probe 1 showed appreciable fluorescence response which is presumably because of the initiation of the aromatic nucleophilic substitution reaction in the presence of excess hydroxide ions to produce back phenol 2 responsible for ESIPT. However, probe 1 could work very efficiently for aromatic thiols at neutral pH and ambient temperature, which would enable the use of the probe in the detection of these pollutants in biological and environmental samples directly.

Fig. 1 Fluorescence response of probe 1 (30 μM) with (1 equiv.) and without thiophenol at different pH (pH 1–9.0) (λ_ex = 365 nm).

3.3 Thiophenol sensing by probe 1: spectrophotometric studies and mechanistic aspect

To understand the sensing characteristic of probe 1 an equivalence study was conducted. In a typical experiment, the fluorescence response of probe 1 upon addition of 0–3 equiv. of thiophenol in 45% DMF–PBS buffer was measured. The solution was incubated for 15 min after addition of each portion of thiophenol before measuring the fluorescence response of the corresponding solution. An intense greenish blue fluorescence was observed at λ_max 462 nm which gradually intensified with increasing concentration of thiophenol (Fig. 2). The fluorescence enhancement at 462 nm was up to 16 fold. The probe responded linearly till addition of 1 equiv. of thiophenol. Upon addition of more amount of thiophenol nominal increase in fluorescence response was seen indicating that the probe is mostly used up by addition of just 1 equiv. of thiophenol. However, 3 equiv. of thiophenol was required to reach the actual saturation point. This indicates that the reaction slows down towards the end and excess thiophenol is required for complete conversion, which is a common phenomenon for most of the nucleophilic substitution reaction.

Fig. 2 Fluorescence response of a probe 1 (30 μM) upon addition of thiophenol (0–90 μM) in 45% DMF : PBS (pH 7.2) [excitation wavelength is 395 nm]. Inset: plot of the increment in emission against no. of equiv. of thiophenol.

To validate the sensing mechanism of probe 1, a relatively large scale reaction was carried out by adding thiophenol to probe 1 under the prevailing condition and the product was isolated and characterized by ¹H NMR and mass spectroscopy. It was observed that the spectra of the product obtained from fluorimetric study exactly match with the spectra of compound 2; thus, confirming that the non-fluorescent probe 1 was converted back to strongly fluorescent compound 2 by thiophenol driven nucleophilic substitution reaction. It is well-established that 2-(2-hydroxyphenyl)benzothiazole (2) shows fluorescence by excited state intramolecular proton transfer (ESIPT). Since the sensing condition use neutral pH the newly liberated benzothiazole derivative (2) remains in the protonated form. The internal H-transfer occurs from phenolic –OH to –N– of benzothiazole moiety of the molecule in the excited state which causes large electron delocalization from one aromatic ring to other and the molecule becomes fluorescence active. The above study clearly indicated that probe 1 is highly useful for the detection of aromatic thiols by spontaneous “ESIPT”.

3.4 Selectivity of aromatic thiols over other thiols

To confirm the selectivity of probe 1 towards thiophenol, it was treated with a broad variety of species under the standard condition and corresponding fluorescence response was recorded. The probe was separately treated with several L-amino acids such as glycine, alanine, leucine, argenine, histidine, proline, and thiol containing cysteine and glutathione. This study also included an aliphatic thiol, 2-mercaptoethanol, and aromatic competing nucleophiles phenol and aniline. It revealed that each of the above mentioned species educed very nominal fluorescence response indicating that the probe is inactive to aliphatic thiols and nucleophiles under the prevailing reaction condition (Fig. 3 and S1 of ESI†). The same study was extended to other aromatic thiols such as 2-aminothiophenol, 4-aminothiophenol, pyridine-2-thiol, 4-bromothiophenol and 4-nitrothiophenol. It was observed that 1 could efficiently sense the presence of most of the aromatic thiols showing a profound fluorescence response. However, probe 1 was found to be inactive to 4-nitrothiophenol indicating its inefficacy for the detection of aromatic thiols having strong electron withdrawing groups in the ring (Fig. 3 and S1 of ESI†). This is an expected outcome because the nucleophilicity of aromatic-SH group is significantly reduced for 4-nitrothiophenol and therefore, the probe does not undergo S_NAr reaction at room temperature. In a separate competitive experiment, the probe was exposed to 1 equiv. of thiophenol in the presence 5 equiv. each of phenol, aniline and 2-mercaptoethanol and corresponding fluorescence response was measured. As showed in Fig. 3 the presence of other nucleophile could cause little effect in spontaneous detection of thiophenol by probe 1.

Fig. 3 Maximum fluorescence response of probe 1 in 45% DMF–PBS (pH = 7.2) upon addition of different amino acids, aliphatic thiols, other nucleophiles (1 = blank; 2 = cysteine, 3 = argenine, 4 = leucine, 5 = L-proline, 6 = alanine, 7 = glycine, 8 = histidine, 9 = glutathione, 10 = phenol, 11 = aniline, 12 = 2-mercaptoethanol, 13 = HSO₃⁻, 14 = thiophenol, 15 = pyridine-2-thiol, 16 = 2-aminothiophenol, 17 = 4-aminothiophenol, 18 = 4-bromothiophenol and 19 = thiophenol in the presence of 5 equiv. of other nucleophiles) in the presence of a fixed concentration of probe 1 (30 μM) [excitation wavelength = 365 nm].

3.5 Limit of detection

The limit of detection (LOD) is one of the most important properties of any probe that needs to be assessed to understand potential applicability of the probe as an analytical tool for real sample analysis. It was observed that probe 1 is well verge to detect trace level of thiophenols in water. Under the standard condition probe 1 was found to respond to thiophenols below micro molar concentration range (from 3 × 10⁻⁸ M of thiophenol) and the linearity was obtained from 9 × 10⁻⁸ M of thiophenol. Thus, the detection limit (LOD) and limit of quantification (LOQ) of probe 1 was considered as 3 × 10⁻⁸ M (3.3 ppb) and 9 × 10⁻⁸ M (10 ppb), respectively (Fig. 4).

Fig. 4 A fluorescence intensity plot of the probe 1 against a low concentration range of thiophenol. The straight line was obtained from 9 × 10⁻⁸ M of thiophenol (λ_ex = 365 nm).

3.6 Real sample analysis

Probe 1 could express its potential applicability as an efficient analytical tool in the detection of thiophenols especially in the environment. Therefore, probe 1 was applied for qualitative/quantitative detection of thiophenol levels in several real samples.

3.6.1 Detection of thiophenol in water samples. To demonstrate the applicability of this probe for monitoring the contamination of thiophenol in industrial effluent, a proof-of-concept experiment was performed on water samples pre-spiked with thiophenol. Thus, water samples (1 mL) were collected from tap and river, filtered through microfiltration membrane and then spiked with known quantities of thiophenol (1 mM) in DMF and marked as sample A and B. To these samples, DMF and PBS were added to make the final concentrations of thiophenol as 1.5 μM for sample A and 3.0 μM for sample B in 45% DMF–PBS solution (pH 7.2). These samples were further treated with probe 1 (30 μM). After 15 min of incubation at room temperature, the fluorescence responses of the solutions were recorded and the intensity at 462 nm was plotted onto the standard curve. As shown in Fig. 5, our analytic tool worked efficiently in the detection of pre-spiked thiophenol in water samples with high recovery (97–100.2%) [see ESI, Table S1†].

Fig. 5 Maximum fluorescence response of probe 1 towards the real samples spiked with thiophenol (λ_ex = 365 nm).

3.6.2 Detection of thiophenol vapour by paper strips and TLC plates. As thiophenol is volatile liquid and the resulting vapor can cause severe health issues like headache and irritation, it is of immense importance to detect its vapors in the atmosphere. The paper strip of probe 1 was prepared by soaking it in a solution of probe 1 in DMF and then kept in a chemical storage cabinet for 15 min where an uncapped bottle of thiophenol was preplaced. As a control experiment one more paper strip was dipped in the same probe solution and placed in storage cabinet without thiophenol. The whole strip, which was placed in the cabinet with uncapped bottle of thiophenol, started glowing under exposure of long wavelength UV light (365 nm), in contrast, no fluorescence was seen from the other paper strip (Fig. 6). Similarly, probe 1 modified TLC plate showed intense blue fluorescence spot under long wavelength UV light upon exposure to thiophenol for sometimes. Therefore, it can be concluded that the vapours of thiophenol could spontaneously react with probe 1 even on paper strip or TLC plate, and converts it back to the highly fluorescent compound (2). In a similar manner the TLC plates and paper strips were also employed for the selective detection of aqueous thiophenol. For this purpose the aqueous solution of thiophenols and other analytes were spotted over TLC plates at the same area where the probe 1 were spotted. Similarly, the paper strips soaked with probe 1 was dipped in aqueous solution of respective analytes. In both the cases only the solution containing thiophenol showed bright fluorescence under long wavelength UV light (354 nm) indicating that the same technique is equally applicable for the detection of thiophenols in the solution phase as well (see ESI, Fig. S2†).

Fig. 6 (i) Showing the effect of vapors of aromatic thiols on probe 1 before and after exposure to thiophenol on TLC plate and (ii) showing the effect of vapors of aromatic thiols on the paper strips spiked with probe 1 ((A) spiked with probe 1, (B) spiked with probe 1 and kept in a cabinet containing open bottle of thiophenol).

3.6.3 Detection of thiophenol in soil samples. To examine whether the probe can detect thiophenol contamination in soil probe 1 was used for the estimation of pre-spiked thiophenol in a soil sample. In a typical experiment, 3 mL of probe 1 (30 μM) in 45% DMF–PBS (pH 7.2) was added to soil sample (90 mg) pre-spiked with known quantity thiophenol (1.2 μM) in ethanol. This sample was mixed thoroughly, incubated at room temperature for 15 min, after which the solution was filtered for fluorescence measurement. Again, the concentration of thiophenol was cross-examined by plotting the fluorescence intensity value on the standard curve. A near equal level of concentrations of thiophenol indicated that the probe is equally effective in determination of aromatic thiols in the soil samples as well.

3.7 Cytotoxicity and fluorescent cell imaging

Another potential application of probe 1 could be in the detection of the thiophenols in living cells. For this purpose, the cytotoxicity of probe 1 was first examined and it was used for the detection of aromatic thiols in thiophenol contaminated HeLa cells.

3.7.1 Cytotoxicity assay. As a part of the standard protocol the cytotoxicity of probe 1 was assessed on HeLa cell line using MTT assay before fluorescent cell imaging studies. In the concentration range 1–20 μM of probe 1 hardly any cell death was seen after 24 h of incubation indicating the probe molecule is non-toxic even at much higher concentration than that required for the detection studies (see ESI, Fig. S3†).

3.7.2 Fluorescence imaging of living cells. The usefulness of probe 1 for fluorescence imaging of thiophenols in the living cells was investigated. Therefore, HeLa cells were pre-treated with thiophenol (1 μM) in a growth medium and then incubated with probe 1 (1 μM) for 30 min. A bright fluorescence was observed when the cell-line was placed under a Nikon eclipse TS100 microscope (Fig. 7g). By contrast, in a control experiment, incubation of HeLa cells with only probe 1 provided no significant fluorescence (Fig. 7e). These observations indicate that probe 1 is cell membrane permeable and able to interact with the intercellular thiophenol as well to produce fluorescence signals. In a separate control experiment, the HeLa cells were pre-treated with thiophenol (1 μM), then incubated with N-ethylmaleimide (1 μM, as a thiol scavenger), and then treated with probe 1 (1 μM). As expected, no marked fluorescence signal was observed from cells (Fig. 7h), indicating the selective reaction of probe 1 with thiophenol. These preliminary studies revealed that probe 1 is useful for in vitro imaging to monitor thiophenols in biological samples as well.

Fig. 7 Detection of thiophenol in HeLa cells with probe 1. Phase contrast (a–d) and fluorescence images (e–h) of the HeLa cells after incubating with probe 1 (1 μM), thiophenol (1 μM), probe 1 (1 μM) in presence of thiophenol (1 μM), probe 1 in presence of thiophenol (1 μM) and N-ethylmaleimide (1 μM), respectively.

4. Conclusion

In summary, we have rationally designed a novel, “turn-on” type fluorimetric sensor for the detection of aromatic thiols in aqueous systems. The probe was synthesized in high yield via a one-pot method in water maintaining environment friendly conditions all throughout. The function of the probe is based on simple thiol driven cleavage of the dinitrophenyl ether linkage of probe 1 to release fluorescent 2-(2-hydroxyphenyl)benzothiazole (2). The probe utilizes strong nucleophilic character of aromatic thiols for their selective detection among many other interfering species including thiol containing amino acids. Probe 1 is effective for thiophenol (PhSH) and other aromatic thiols with electron donating substituents but inactive to aromatic thiols with strong electron withdrawing substituents (e.g. 4-nitrothiophenol). The advantage of this probe is that it shows excellent results under neutral condition at room temperature facilitating its wide scope of applications at physiological pH. Notably, the probe is found to be capable of detecting pre-spiked thiophenols in water, soil and living cells. We presume, the probe with green synthetic profile, cost effective single-step synthesis, high selectivity and sensitivity, fast signal transduction and low limit of detection (3 × 10⁻⁸ M) would certainly be a potential candidate for practical applications.

Acknowledgements

A. C. thanks DST (India) (project no. SR/FT/CS-092/2009) for financial support. M. B. thanks CSIR (India) (project No. 02(0075)/12/EMR-II) for research grant. D. G. K. is indebted to DST (India) for fellowship.

Notes and references

1. (a) K.-M. Roy, in Ullmann's Encyclopaedia of Industrial Chemistry, Wiley, New York, 7th edn, 2007; (b) J. C. Love, L. A. Estroff, J. K. Kriebel, R. G. Nuzzo and G. M. Whitesides, Chem. Rev., 2005, 105, 1103–1170.
2. E. Bingham, B. Cohrssen and C. H. Powell, in Patty's Industrial Hygiene and Toxicology, John Wiley & Sons, Inc, New York, 5th edn, 2001, p. 722.
3. (a) T. P. Heil and R. C. Lindsay, J. Environ. Sci. Health, Part B, 1989, 24, 349–360; (b) E. J. Fairchild and H. E. Stokinger, Am. Ind. Hyg. Assoc. J., 1958, 19, 171–188.
4. (a) R. Munday, Free Radical Biol. Med., 1989, 7, 659–673; (b) T. P. Heil and R. C. Lindsay, J. Environ. Sci. Health, Part B, 1989, 24, 349–360; (c) P. Amrolia, S. G. Sullivan, A. Stern and R. Munday, J. Appl. Toxicol., 1989, 9, 113–118.
5. (a) R. M. Duke, E. B. Veale, F. M. Pfeffer, P. E. Kruger and T. Gunnlaugsson, Chem. Soc. Rev., 2010, 39, 3936–3953; (b) M. J. Culzoni, A. M. de la Pena, A. Machuca, H. C. Goicoechea and R. Babiano, Anal. Methods, 2013, 5, 30–49; (c) M. E. Jun, B. Roy and K. H. Ahn, Chem. Commun., 2011, 47, 7583–7601; (d) J. Chan, S. C. Dodanil and C. J. Chang, Nat. Chem., 2012, 4, 973–984; (e) X. Chen, G. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2012, 41, 4610–4630.
6. (a) S. Jung, X. Chen, J. S. Kim and J. Yoon, Chem. Soc. Rev., 2013, 42, 6019–6031; (b) X. Chen, Y. Zhaou, X. Peng and J. Yoon, Chem. Soc. Rev., 2010, 39, 2120–2135; (c) H. Peng, W. Chen, Y. Cheng, L. Hakuna, R. Strongin and B. Wang, Sensors, 2012, 12, 15907–15946; (d) C. Yin, F. Huo, J. Zhang, R. Martínez-Máñez, Y. Yang, H. Lv and S. Li, Chem. Soc. Rev., 2013, 42, 6032–6059.
7. For selected recent references see: (a) B. Tang, Y. Xing, P. Li, N. Zhang, F. Yu and G. Yang, J. Am. Chem. Soc., 2007, 129, 11666–11667; (b) X. Chen, S.-K. Ko, M. J. Kim, I. Shin and J. Yoon, Chem. Commun., 2010, 46, 2751–2753; (c) V. Hong, A. A. Kislukhin and M. G. Finn, J. Am. Chem. Soc., 2009, 131, 9986–9994; (d) M. H. Lee, J. H. Han, P.-S. Kwon, S. Bhuniya, J. Y. Kim, J. L. Sessler, C. Kang and J. S. Kim, J. Am. Chem. Soc., 2012, 134, 1316–1322; (e) Y.-Q. Sun, M. Chen, J. Liu, X. Lv, J.-F. Li and W. Guo, Chem. Commun., 2011, 47, 11029–11031; (f) M.-Y. Jia, L.-Y. Niu, Y. Zhang, Q.-Z. Yang, C.-H. Tung, Y.-F. Guan and L. Feng, ACS Appl. Mater. Interfaces, 2015, 7, 5907–5914; (g) L. Wang, T. Yao, S. Shi, Y. Cao and W. Sun, Sci. Rep., 2014, 4, 5320,  DOI:10.1038/srep05320; (h) Z. Chen, D. Lu, Z. Cai, C. Dong and S. Shuang, Luminescence, 2014, 29, 722–727; (i) L. Yi, H. Li, L. Sun, L. Liu, C. Zhang and Z. Xi, Angew. Chem., Int. Ed., 2009, 48, 4034–4037; (j) Y. Li, Y. Duan, J. Li, J. Zheng, H. Yu and R. Yang, Anal. Chem., 2012, 84, 4732–4738; (k) Y. Yue, C. Yin, F. Huo, J. Chao and Y. Zhang, Sens. Actuators, B, 2016, 223, 496–500; (l) F. J. Huo, Y. Q. Sun, J. Su, J. B. Chao, H. J. Zhi and C. X. Yin, Org. Lett., 2009, 11, 4918–4921; (m) Y. Yang, F. Huo, C. Yin, A. Zheng, J. Chao, Y. Li, Z. Nie and R. Martínez-Máñez, Biosens. Bioelectron., 2013, 47, 300–306.
8. (a) J. Bouffard, Y. Kim, T. M. Swager, R. Weissleder and S. A. Hilderbrand, Org. Lett., 2008, 10, 37–40; (b) H. Guo, Y. Jing, X. Yuan, S. Ji, J. Zhao, X. Li and Y. Kan, Org. Biomol. Chem., 2011, 9, 3844–3853; (c) X.-D. Jiang, J. Zhang, X. Shao and W. Zhao, Org. Biomol. Chem., 2012, 10, 1966–1968; (d) Z. Wang, D.-M. Han, W.-P. Jia, Q.-Z. Zhou and W.-P. Deng, Anal. Chem., 2012, 84, 4915–4920; (e) W. Sun, W. Li, J. Li, J. Zhang, L. Du and M. Li, Tetrahedron Lett., 2012, 53, 2332–2335; (f) X. Shao, R. Kang, Y. Zhang, Z. Huang, F. Peng, J. Zhang, Y. Wang, F. Pan, W. Zhang and W. Zhao, Anal. Chem., 2015, 87, 399–405; (g) D. Kand, P. S. Mandal, T. Saha and P. Talukdar, RSC Adv., 2014, 4, 59579–59586.
9. (a) J. Li, C.-F. Zhang, S.-H. Yang, W.-C. Yang and G.-F. Yang, Anal. Chem., 2014, 86, 3037–3042; (b) S. K. Malwal, A. Labade, A. S. Andhalkar, K. Sengupta and H. Chakrapani, Chem. Commun., 2014, 50, 11533–11535; (c) D. Kand, P. K. Mishra, T. Saha, M. Lahiri and P. Talukdar, Analyst, 2012, 137, 3921–3924; (d) W. Lin, L. Long and W. Tan, Chem. Commun., 2010, 46, 1503–1505; (e) W. Jiang, Q. Fu, H. Fan, J. Ho and W. Wang, Angew. Chem., Int. Ed., 2007, 46, 8445–8448; (f) X.-L. Liu, X.-Y. Duan, X.-J. Du and Q.-H. Song, Chem.–Asian J., 2012, 7, 2696–2702; (g) X. Liu, F. Qi, Y. Su, W. Chen, L. Yang and X. Song, J. Mater. Chem. C, 2016, 4, 4320–4326; (h) W. Zhao, W. Liu, J. Ge, J. Wu, W. Zhang, X. Meng and P. Wang, J. Mater. Chem., 2011, 21, 13561–13568.
10. W. Chen, Y. Zhao, T. Seefeldt and X. Guan, J. Pharm. Biomed. Anal., 2008, 48, 1375–1380.
11. T. Wang, E. Chamberlain, H. Shi, C. D. Adams and Y. Ma, Int. J. Environ. Anal. Chem., 2010, 90, 948–961.
12. O. Pluchery, C. Humbert, M. Valamanesh, E. Lacaze and B. Busson, Phys. Chem. Chem. Phys., 2009, 11, 7729–7737.
13. J. Onuegbu, A. Fu, O. Glembocki, S. Pokes, D. Alexson and C. M. Hosten, Spectrochim. Acta, Part A, 2011, 79, 456–460.
14. (a) J. C. Warner, A. S. Cannon and K. M. Dye, Environ. Impact Assess. Rev., 2004, 24, 775–799; (b) R. A. Sheldon, Green Chem., 2005, 7, 267–278; (c) F. Kerton and R. Marriott, in Alternative Solvents for Green Chemistry, RSC Publishing, 2nd edn, 2013.
15. X. Li, X. Gao, W. Shi and H. Ma, Chem. Rev., 2014, 114, 590–659.
16. (a) D. G. Khandare, H. Joshi, M. Banerjee, M. Majik and A. Chatterjee, Anal. Chem., 2015, 87, 10871–10877; (b) A. Chatterjee, D. G. Khandare, P. Saini, A. Chattopadhyay, M. S. Majik and M. Banerjee, RSC Adv., 2015, 5, 31479–31484; (c) D. G. Khandare, H. Joshi, M. Banerjee, M. S. Majik and A. Chatterjee, RSC Adv., 2014, 4, 47076–47080; (d) D. G. Khandare, V. Kumar, A. Chattopadhyay, M. Banerjee and A. Chatterjee, RSC Adv., 2013, 3, 16981–16985; (e) S. Hazra, S. Balaji, M. Banerjee, A. Ganguly, N. N. Ghosh and A. Chatterjee, Anal. Methods, 2014, 6, 3784–3790; (f) V. Kumar, M. Banerjee and A. Chatterjee, Talanta, 2012, 99, 610–615.
17. V. S. Padalkar and S. Seki, Chem. Soc. Rev., 2016, 45, 169–202 , and references cited there in.

---

Footnote

† Electronic supplementary information (ESI) available: Synthetic procedure of compound 2; real sample analysis data; scanned copies of NMR and HR-MS spectra of 1 and 2. See DOI: 10.1039/c6ra07046a

---