1 Introduction

Reactive oxygen species (ROS) such as \(^{1}\hbox {O}_{{2}}\), \(^{\bullet }\hbox {OH}\), \(\hbox {O}_{{2}}^{-}\), \(\hbox {H}_{{2}}\hbox {O}_{{2}}\), t-BuOOH and HOCl/\(\hbox {ClO}^{-}\) cause oxidative stress and play vital roles in toxicology as well as pathology.[1, 2] Reactive oxygen species (ROS) have very close involvement in diverse biological processes including its alleged role in many diseases like cancer and neurodegenerative disorders.[3,4,5,6] Hypochlorous acid (HClO) or its conjugate base hypochlorite (ClO\(^-\)) is a well-known ROS which is commonly used as bleaching agents.[7,8,9] Hypochlorous acid (HClO)/hypochlorite (ClO\(^-\)) can be generated endogenously from the peroxidation reaction of hydrogen peroxide and chloride ion, catalyzed by the enzyme myeloperoxidase (MPO).[10,11,12,13,14] Importantly, Hypochlorous acid (HClO) can serve as a powerful antimicrobial agent and it exerts a destructive effect on invading bacteria and pathogens to enable a defence mechanism in the immune system.[15, 16] However, unregulated ClO\(^-\) production could lead to the damage of DNA and proteins which might induce several ailments such as cardiovascular diseases, atherosclerosis, osteoarthritis, rheumatoid arthritis, lung injury and even various types of cancer.[17,18,19,20,21,22,23] Hence, the sensitive detection of ClO- with high selectivity is much awaited to understand the biological insight of ClO\(^-\) related diseases.

The fluorescence-based sensing probes[24,25,26,27,28,29,30,31,32,33] are much more attractive compared to other systems owing to the superiority of fluorescence technique in terms of high sensitivity, rapid response and user friendliness along with its ability to provide non-invasive temporal detection of target analytes in living cells. However, till now there are only a few reports which deal with the sensitive detection of (HClO)/hypochlorite (ClO\(^-\)) using fluorescent probe.[5, 34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49] Some mitochondria-targeted fluorescent probes for HClO/ClO\(^-\) have been reported recently which significantly contribute to research related to the role of ClO\(^-\) in physiology and pathology.[50,51,52,53,54,55] Among them, a number of reported fluorescent probes for (HClO)/hypochlorite (ClO\(^-\)) detection are mostly confined to oxime and imine based probes.[5, 34,35,36,37,38,39,40,41,42,43,44,45,46, 52] But oxime or imine based probes might be vulnerable to acid catalyzed hydrolysis which clearly may curb their application potential in complex biological milieu.[19] Especially inside the cancer cells, wherein the lower pH environment exists, the stability of oxime-based fluorescent probe might become a pertinent issue.[56,57,58,59,60] Hence, cyanine based probes[61, 62] with high stability could be an alternative system to develop hypochlorite sensor.

In this context, we would like to report a new cyanine based spiropyran fluorogenic probe which can demonstrate a highly selective TURN-ON fluorescence response towards hypochlorite (ClO\(^-\)) in 1:1 methanol-aqueous medium. The sensing process was explained because of the disruption in extended conjugation due to the addition reaction between L and ClO- anion.

2 Experimental

2.1 General information and materials

All the materials for synthesis were purchased from commercial suppliers and used without further purification. The absorption spectra were recorded on a Perkin-Elmer Lamda-25 UV-Vis spectrophotometer using 10 mm path length quartz cuvettes in the range of 300–800 nm wavelength, whereas fluorescence measurements were performed on a Horiba Fluoromax-4 spectrofluorometer using 10 mm path length quartz cuvettes with a slit width of 3 nm at 298 K. The mass spectrum of the probe L was obtained using Waters Q-ToF Premier mass spectrometer. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 600 MHz NMR instrument. The chemical shifts were recorded in parts per million (ppm) on the scale. The following abbreviations are used to describe spin multiplicities in \(^{1}\hbox {H}\) NMR spectra: s \(=\) singlet; d \(=\) doublet; t \(=\) triplet; q \(=\) quartet, m \(=\) multiplet.

2.2 Synthesis of the probe L

First, 3–ethyl–1,1,2–trimethyl–1H–benzo[e]indol–3–ium was synthesized through the reported procedure as described in our previously published work (first step of Scheme 1).[26, 28] Thereafter, 1.0 mmol of this 3–ethyl–1,1,2–trimethyl–1H–benzo[e]indol–3–ium was taken in a round-bottomed flask and it was dissolved in EtOH by gentle warming. 1.5 mmol of anhydrous sodium acetate was then added to it and the mixture was refluxed for 1 h. Subsequently, 1.1 mmol of 2-hydroxy-1-naphthaldehyde was added to the reaction mixture in the heating condition and it was further refluxed for 6 h (Scheme 1). A colourless crystalline solid product was obtained upon cooling the reaction mixture to the room temperature. The solid product was then filtered, washed thoroughly with cold methanol and dried in a desiccator. Calculated yield: 64%. \(^{1}\hbox {H}\) NMR [600 MHz, \(\hbox {CDCl}_{{3}}\), TMS, J (Hz), \(\delta \) (ppm)]: 8.06 (1H, d, \(J = 8.4\)), 7.95 (1H, d, \(J = 8.4\)), 7.81 (1H, d, \(J = 8.4\)), 7.76 (2H, m), 7.65 (2H, m), 7.53 (1H, t, \(J = 7.2\)), 7.41 (1H, t, \(J = 7.2\)), 7.35 (1H, t, \(J = 7.2\)), 7.22 (1H, t, \(J = 7.2\)), 7.02 (1H, d, \(J=9.0\)), 6.93 (1H, d, \(J=9.0\)), 5.87 (1H, t, \(J = 10.2\)), 3.46 (2H, m), 1.69 (3H, s), 1.37 (3H, s) 1.22 (3H, t, \(J = 7.2\)). \(^{13}\hbox {C}\) NMR [150 MHz, \(\hbox {CDCl}_{{3}}\), TMS, \(\delta \) (ppm)]: 152.80, 145.13, 130.46, 130.18, 129.99, 129.67, 129.21, 128.98, 128.81, 128.77, 126.85, 126.34, 125.25, 123.34, 121.45, 121.39, 120.70, 117.90, 117.68, 110.23, 110.08, 105.81, 53.86, 38.05, 24.16, 21.82, 14.93. ESI-MS (positive mode, m/z) Calculated for \(\hbox {C}_{{28}}\hbox {H}_{{25}}\hbox {NO}\) \((\hbox {M}+\hbox {H}^{+})\): 392.2009. Found: 392.2051.

Scheme 1
scheme 1

Design and synthesis of probe L.

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2.3 Preparation of ROS and RNS

Various ROS and RNS including \(^{1}\hbox {O}_{{2}}\), \(^{\bullet }\hbox {OH}\), \(\hbox {O}_{{2}}^{-}\), \(\hbox {H}_{{2}}\hbox {O}_{{2}}\), t-BuOOH, \(\hbox {ClO}^{-}\), \(\hbox {NO}_{{2}}^{-}\), \(\hbox {NO}_{{3}}^{-}\), NO and \(\hbox {ONOO}^{-}\) were prepared according to the following procedures. The exact concentration of hypochlorite (\(\hbox {OCl}^{-})\) solution was determined from the extinction coefficient of 350 \(\hbox {M}^{-1}\hbox {cm}^{-1}\) (292 nm) at pH=9.0. Hydroxyl radical (\({\bullet }\hbox {OH}\)) was produced using Fenton reaction, wherein \(\hbox {Fe}(\hbox {NH}_{4})_{2}(\hbox {SO}_{4})_{2}\cdot \hbox {6H}_{{2}}\hbox {O}\) was reacted with 10 equivalents of \(\hbox {H}_{{2}}\hbox {O}_{{2}}\) to generate \({\bullet }\hbox {OH}\). The concentration of the prepared \({\bullet }\hbox {OH}\) was estimated from the concentration of \(\hbox {Fe}^{2+}\). Commercially available stock of \(\hbox {H}_{{2}}\hbox {O}_{{2}}\) was used for the study and concentration of the \(\hbox {H}_{{2}}\hbox {O}_{2}\) solution was estimated by optical absorbance at 240 nm. Singlet oxygen \((^{1}\hbox {O}_{2})\) was generated by adding NaOCl to \(\hbox {H}_{{2}}\hbox {O}_{{2}}\) according to the reported procedure.[63] The source of \(\hbox {NO}_{{2}}^{-}\) and \(\hbox {NO}_{{3}}^{-}\) were \(\hbox {NaNO}_{2}\) and \(\hbox {NaNO}_{{3}}\) respectively. Nitric oxide (NO) was prepared from sodium nitro-ferricyanide(III) dihydrate. Peroxynitrite (ONOO\(^-\)) was prepared following the reported method[64] and the concentration of it was estimated by using an extinction coefficient of 1670 \(\hbox {M}^{-1}\hbox {cm}^{-1}\) (302 nm).[65] Superoxide (\(\hbox {O}_{{2}}^{-})\) is prepared from \(\hbox {KO}_{{2}}\). t-BuOOH was obtained from commercial suppliers.

2.4 UV-Vis and fluorescence spectroscopic studies

Stock solutions of various anions (\(1 \times 10^{-1} \hbox { mol.L}^{-1})\) were prepared in methanol or Millipore water depending upon the preferred solubility of the analytes chosen. A stock solution of L (\(5 \times 10^{-3}\hbox { mol.L}^{-1})\) was prepared in DMSO. The solution of L was then diluted to \(10 \times 10^{-6}\hbox { mol.L}^{-1}\) for spectral studies by taking only \(4.0\, \upmu \hbox {L}\) stock solution of L and making the final volume to 2.0 mL by adding 1:1 methanol-water mixed solvent. In fluorescence selectivity experiment, the test samples were prepared by placing appropriate amounts of the stock solutions of the respective anions/analytes into 2.0 mL of probe solution (\(10 \times 10^{-6}\hbox { mol.} \hbox {L}^{-1})\). For UV-visible and fluorescence titration experiments, another set of \(\hbox {OCl}^{-}\) standard solution having 5.0 mM concentration was prepared by diluting the earlier prepared stock solutions (\(1 \times 10^{-1}\hbox { mol.L}^{-1})\) in Millipore water. Quartz optical cells of 1.0 cm path length were filled with 2.0 mL solutions of L for UV-visible and fluorescence titration experiments respectively, to which the newly prepared stock solutions of \(\hbox {OCl}^{-}\) (5.0 mM) were gradually added using a micropipette as necessary. For fluorescence measurements, excitation was provided at 360 nm and emission was acquired from 380 nm to 650 nm. Spectral data were recorded within 1 min after addition of the analytes except for kinetic studies.

2.5 Detection limit

The detection limit was calculated on the basis of the fluorescence titration. The fluorescence emission spectrum of L was measured 10 times, and the standard deviation of blank measurement was estimated (\(\uplambda _{\mathrm{em}} = 435\hbox { nm}\)). To measure the slope, the fluorescence emission at 435 nm was plotted as a function of the concentration of \(\hbox {OCl}^{-}\) from the titration experiment. The detection limit was then calculated using the following equation:

$$\begin{aligned} \hbox {Detection limit} = 3\upsigma /\hbox {k} \end{aligned}$$
(1)

Where \(\upsigma \) is the standard deviation of blank measurement, and k is the slope between the fluorescence emission intensity versus [\(\hbox {OCl}^{-}\)].

2.6 Crystallization of L

A small amount of brown-red solid of L was taken in a test tube containing DCM-EtOH mixture and sonicated for 5 min to obtain a clear solution. Then the solution was allowed to evaporate slowly at room temperature and after one week, X-ray mountable plate-shaped orange-red colored crystals appeared at the bottom of the test tube.

2.6.1 Crystallographic refinement details

Herein, a suitable crystal was selected from the mother liquor and immersed into silicone oil, then mounted on the tip of glass fibre and cemented using epoxy resin. Intensity data for all crystals were collected \(\hbox {Mo-K}\upalpha \) radiation (\(\uplambda = 0.71073{\AA }\)) at 298(2) K, with increasing \(\upomega \) (width of 0.3 \(^{\circ }\) per frame) at a scan speed of 6 s/ frame on a Bruker SMART APEX diffractometer equipped with CCD area detector. The data integration and reduction were processed with SAINT[66] software. Empirical absorption correction was applied to the collected reflections with SADABS.[67] The structures were solved by direct methods using SHELXTL-2014 and were refined on F2 by the full-matrix least-squares technique using the SHELXL-2014 program package.[68] Graphics are generated using MERCURY 2.3.[69] In all the cases, non-hydrogen atoms are treated anisotropically. The hydrogen atoms are located on a difference Fourier map and refined. In other cases, the hydrogen atoms are geometrically fixed.

CCDC number – 1572275, formula – \(\hbox {C}_{{28}}\hbox {H}_{{25}}\hbox {NO}\), Fw – 391.49, crystal system – monoclinic, space group – P 21/c, \(a = 22.441(4)\) Å, \(b = 11.222\) (14) Å, \(c = 18.966(2)\) Å, \(\alpha = 90.00\), \(\beta = 114.981(4)\), \(\gamma = 90.00\), \(V = 4329.5(10)\) Å\(^{3}\), \(Z = 4\), \(D_{\mathrm{c}} = 0.601\hbox { g cm}^{-3}\), \(\mu \) \((\hbox {Mo K}\upalpha ) = 0.036 \hbox { mm}^{-1}\), \(F(000) = 832.0\), \(T = 298(2)\hbox { K}\), \(\hbox {Theta}_{\mathrm{max}} = 28.532\), total no. of reflections \( = 75550\), independent reflections \(=\) 10726, observed reflections \( = 8457\), parameters refined \(=\) 274, \(R_{1}\), \(I > 2\upsigma \hbox {(I)} = 0.1438\), \(\hbox {w}R_{2}\), \(I > 2\upsigma \hbox {(I)} = 0.3210\), GOF (\(F^{2}) = 0.993\).

3 Results and Discussions

3.1 Design, synthesis and structural elucidation of the probe L

The probe was synthesized by following a procedure summarized in Scheme 1. Condensation of an indole derivative and 2-hydroxy-1-naphthaldehyde in the presence of anhydrous sodium acetate led to the formation of the probe L with about 64% product yield. The synthesized probe was well-characterized by NMR and MS analysis. The structure of L was further reaffirmed by its crystal structure analysis. The X-ray crystal structure reveals that L exists in spiropyran (SP) ring form rather than merocyanine (MC) form in the solid state. The X-ray single-crystal structure of L showed that it has adopted a non-centrosymmetric molecular arrangement with the polar space group of P 21/c (experimental methods). The crystal packing of the probe L suggested that two different types of conformers are present in a unit cell of L (Figure 1a). Both the types of conformers of L are subjected to eleven short-range \(\hbox {C}-\hbox {H}\cdots \uppi \) intermolecular interactions each (Figure 1b–1c). The aromatic naphthyl planes of the two conformers are found to have almost perpendicular orientations (87.7 \(^{\circ })\) (Figure 1d). The packing diagrams of L have been presented in Figure 1e and Figure 1f.

Fig. 1
figure 1

(A) ORTEP molecular structure at 30% probability level of two conformers, Intermolecular \(\hbox {C}-\hbox {H}\cdots \uppi \) interactions within the range of 2.814–2.899 Å in (B) conformer 1, (C) conformer 2, (D) Almost perpendicular orientations of aromatic naphthyl planes of two conformer, (E) Packing motif along crystallographic b axis depicting wave-like architecture construction by alternative conformer array in symmetry-equivalence (blue-conformer 1, green-conformer 2) and (F) molecular packing in single crystals in symmetry-equivalence as viewed along crystallographic c axis.

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3.2 Isomerization of the probe L

It is well known that spiropyran derivatives can exhibit photoisomerization.[70, 71] In the absence of UV irradiation, these types of compounds mainly exist in spiropyran (SP) form and do not show any prominent absorbance in the visible region (Scheme 2). On the contrary, upon UV irradiation it transforms into a merocyanine (MC) form which displays a well-defined absorbance maximum in the visible region.[72] In accordance with this, we found that probe L does not reveal any noticeable absorbance peak in an aqueous medium which clearly indicates that the probe is in the SP form. However, it was noted that when we varied the polarity of the medium by changing the water fraction of a methanol-water mixture, there were interesting changes in the colourimetric behaviour of L (Figure 2). It was observed that when the water fraction of the methanol-water mixture is high (>70%) the probe does not reveal any absorbance peak above 400 nm (Figure 2). However, increasing the methanol content of the mixed solvent leads to the generation of a new absorbance peak at around 583 nm. Interestingly when the methanol content of the mixed solvent is 50% the absorbance maximum of the probe L at 583 nm increases substantially and reaches the highest absorbance value (Figure 2). However, a further increase in the methanol content from 50% to 100% resulted in the gradual decrease in the absorbance maxima at 583 nm (Figure 2). Hence, the study clearly indicates that the probe preferentially attains merocyanine form when there is 50% methanol and 50% water in a methanol-water mixed solvent. As the merocyanine form of the probe could only comprise a potential nucleophilic site, it is important to carry out the sensing studies of L in an optimal condition wherein the conversion to the MC form from SP form will be maximum. Herein, the probe can be easily transformed into MC form without irradiation of UV light just by changing the water fraction of a methanol-water mixed solvent. Hence, we can utilize the probe for sensing purpose with merely adjusting the ratio of methanol and water in a methanol-water mixed solvent. As the maximum conversion to the MC form is attained at 50% methanol and 50% water in a methanol-water mixed solvent, all the subsequent spectral studies of L regarding its sensing pursuit in 1:1 methanol-aqueous solution were carried out at room temperature.

Scheme 2
scheme 2

Solvent polarity induced isomerization of the probe L for sensing study.

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Fig. 2
figure 2

Changes in the absorbance behavior of L upon varying the water fraction of the methanol-water mixture of the mixed solvent; Bottom: visual changes.

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3.3 UV-Vis spectroscopic study of L

Probe L displayed a strong absorption peak at 583 nm (2800 \(\hbox {M}^{-1}\) \(\hbox {cm}^{-1})\) in 1:1 methanol- aqueous solution at room temperature as shown in Figure 3a. The absorption band at 583 nm is possibly originated from the conjugated cyanine moiety present in L in its MC form and the absorption band might be attributed to the \(\uppi -\uppi *\) charge transfer. Initially, UV–Vis spectroscopic studies of L were carried out by recording the absorption spectral changes upon the addition of hypochlorite and other reactive oxygen/nitrogen species. The addition of excess (60 equivalents) of various ROS/\(\hbox {RNS}\) to L induced a negligible change in the colourimetric behaviour of L except for hypochlorite (Figure S6, Supplementary Information). It was noted that among various ROS/RNS only addition of \(\hbox {OCl}^{-}\) could substantially reduce the absorbance maximum of L (Figure 3a) at 583 nm. This spectral change was accompanied by a visible color change as violet (only L) experimental solution turned into almost colorless (L+ \(\hbox {OCl}^{-})\) solution (Figure 3a inset). No other reactive species was able to yield any substantial change in the spectral behaviour when interacted with L, which clearly indicates the high selectivity of the probe towards \(\hbox {OCl}^{-}\). It was also noted that the incremental addition of \(\hbox {OCl}^{-}\) to L (0–60 equivalents) led to a regular decrease in the absorbance maxima of L at 583 nm (Figure 3). So, the UV-Visible spectral studies suggest that the probe L could exhibit good selectivity towards \(\hbox {OCl}^{-}\). A fluorescence study was followed in detail to understand the analytical prospect of L.

Fig. 3
figure 3

UV-Visible spectra of L (\(10\, \upmu \hbox {M}\)) in presence of varying concentration of \(\hbox {OCl}^{-}\) (0–60 equivalents), INSET: Visual change in the color of the solution of L in presence of \(\hbox {OCl}^{-}\) under daylight.

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Fig. 4
figure 4

Fluorescence spectra of L (\(10\, \upmu \hbox {M}\)) in 1:1 methanol-aqueous solution in presence of excess (40 equivalents) of various anions, ROS, RNS and bioanalytes.

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Fig. 5
figure 5

Fluorescence spectra of L (10 \(\upmu \hbox {M}\)) in the presence of varying concentration of \(\hbox {OCl}^{-}\); INSET: Changes in the emission intensity at 435 nm with the addition of equivalents of \(\hbox {OCl}^{-}\).

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3.4 Fluorescence spectral response of L towards \(\hbox {OCl}^{-}\)

The selectivity, sensitivity and response time of L toward \(\hbox {OCl}^{-}\) were investigated using fluorescence spectroscopy. Free probe L was found to be almost non-fluorescent in 1:1 methanol-aqueous solution wherein the merocyanine (MC) form predominates. Hence, upon excitation at 360 nm, L revealed a very low-intensity emission band around 435 nm (Figure 4). However, the fluorescence intensity of the probe L increased more than 10 folds upon addition of 40 equivalents of \(\hbox {OCl}^{-}\) to L and a well-defined emission maximum was found to emerge at 435 nm (Figure 4). However, negligible changes in fluorescence were observed when L interacted with an excess of other ROS and RNS (Figure 4). Especially, the common interfering ROS/RNS like \(^{1}\hbox {O}_{{2}}\), \(^{\bullet }\hbox {OH}\), \(\hbox {O}_{{2}}^{-}\), \(\hbox {H}_{{2}}\hbox {O}_{{2}}\), t-BuOOH, \(\hbox {NO}_{{3}}^{-}\), \(\hbox {NO}_{{2}}^{-}\) and \(\hbox {ONOO}^{-}\) could not bring any noticeable change in the fluorescence intensity of L when added to the solution of L in the similar experimental conditions. Moreover, it was also found that various common anions and bio-analytes including \(\hbox {F}^{-}\), \(\hbox {Cl}^{-}\), \(\hbox {Br}^{-}\), \(\hbox {I}^{-}\), \(\hbox {CH}_{{3}}\hbox {COO}^{-}\), \(\hbox {H}_{{2}}\hbox {PO}_{{4}}^{-}\), \(\hbox {PF}_{{6}}^{-}\), \(\hbox {HCO}_{{3}}^{-}\), \(\hbox {SO}_{{4}}^{2-}\), \(\hbox {HSO}_{{4}}^{-}\), \(\hbox {ClO}_{{4}}^{-}\), \(\hbox {SCN}^{-}\), \(\hbox {S}_{{2}}\hbox {O}_{{3}}^{2-}\), \(\hbox {S}_{{2}}\hbox {O}_{{8}}^{2-}\), pyrophosphate(PPi), cysteine(Cys), homocysteine(Hcy) and glutathione(GSH) hardly induced any change in the fluorescence behavior of L when added to it. Hence, the probe shows an outstanding selectivity towards \(\hbox {OCl}^{-}\). It is also worth mentioning, some recent literature have reported that reactive nucleophilic species like \(\hbox {SO}_{{2}}\) derivatives and \(\hbox {H}_{{2}}\hbox {S}\) can react with various hemi-cyanine dyes containing nucleophilic reaction site.[31, 33, 73] Hence, it was pertinent to check the selectivity of probe L towards \(\hbox {SO}_{{2}}\) derivatives (\(\hbox {SO}_{{3}}^{2-})\) and \(\hbox {H}_{{2}}\hbox {S}\) derivatives (\(\hbox {HS}^{-})\). It was noted that even the presence of 200 equivalents of \(\hbox {SO}_{{2}}\) derivatives and \(\hbox {H}_{{2}}\hbox {S}\) could not bring any noticeable change to the fluorogenic behaviour of the probe L which further reiterated the high selectivity of the probe towards \(\hbox {OCl}^{-}\) (Figure S7, Supplementary Information). Interestingly, the time-dependent fluorescence study of L revealed that it can produce a noticeable change in the fluorescence intensity within seconds of the addition of \(\hbox {OCl}^{-}\) which clearly underlines the rapid sensing aptitude of the probe (Figure S8, Supplementary Information). Hence, the probe can sense hypochlorite selectively with a rapid TURN-ON fluorescence response. However, it is very important to get a quantitative appraisal of the interaction between \(\hbox {OCl}^{-}\) and the probe L to explore the possibility of using the probe in real samples. Thus, a fluorescence titration experiment was performed with varying \(\hbox {OCl}^{-}\) concentration. It was noted that the gradual incremental addition of \(\hbox {OCl}^{-}\) to L in 1:1 methanol-aqueous solution at room temperature resulted in a sequential increase of the emission intensity at 435 nm as evident from Figure 5. It should be mentioned here that the detection limits for \(\hbox {OCl}^{-}\) was determined from the fluorescence titration experiment. The detection limit for \(\hbox {OCl}^{-}\) was found to be \(3.0 \times 10^{-6}\hbox { M}\) (Figure S9, Supplementary Information). In addition, since the Cyanine is sensitive to pH, and the solution of NaOCl is alkaline, the following investigation study was performed to explore the effect of pH on the emission intensity of L at 435 nm upon \(\hbox {OCl}^{-}\) addition (Figure S10, Supplementary Information).

Scheme 3
scheme 3

Plausible sensing mechanism.

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3.5 Mechanism of L responding to OCl\(^{-}\)

The sensing of the \(\hbox {OCl}^{-}\) by L can be accredited to the nucleophilic addition reaction between L and \(\hbox {OCl}^{-}\). The proposed sensing mechanism is depicted in Scheme 3. Essentially the nucleophilic attack of \(\hbox {OCl}^{-}\) to the nucleophilic site of L resulted in the formation of the product L-OCl adduct which is responsible for the observed change in the optical behaviour of L. It is worth mentioning that the plausible nucleophilic attack of \(\hbox {OCl}^{-}\) led to the rupture in the conjugation in L which is reflected in the observed decolouring of the solution of L upon interacting with \(\hbox {OCl}^{-}\). To validate the proposed sensing mechanism mass spectrometric study was conducted. The mass spectrometric analysis indicated towards the formation of L-OCl adduct when L interacted with \(\hbox {OCl}^{-}\) by showing a prominent peak at \(\hbox {m/z} = 485.6401\) (Figure S11, Supplementary Information). Moreover, a reverse phase HPLC study of L carried out in the presence of \(\hbox {OCl}^{-}\) also further endorsed the formation of L-OCl adduct. It was observed that HPLC of pure L resulted in a single peak with a retention time of 26.93 min. However, when HPLC was run after the addition of excess \(\hbox {OCl}^{-}\) there was a new prominent peak generated with a retention time of 25.67 min apart from the pure ligand peak which is indicative of the formation of L-OCl adduct (Figure S12, Supplementary Information). Hence, these studies supported the premise that nucleophilic addition of \(\hbox {OCl}^{-}\) toward L is key to sensing. However, it was interesting to observe that \(\hbox {SO}_{{3}}^{2-}\), even being a better nucleophile could not produce a similar fluorescence change. This might be explained by the steric effect around the nucleophilic site. Probably owing to the larger size of sulfite compared to hypochlorite, it could not undergo a nucleophilic addition reaction at the specific reaction site.

4 Conclusions

In conclusion, a new fluorogenic probe was successfully designed to demonstrate interesting isomerization. The probe demonstrated a highly selective rapid TURN-ON fluorescence response towards hypochlorite in a mixed aqueous medium. The sensing process was explained as a consequence of the probe-OCl adduct formation; which in turn restricted the donor-acceptor extended conjugation to facilitate the TURN-ON fluorescence response. The sensing mechanism is well-corroborated by mass spectroscopic and HPLC study.