A reaction based colorimetric as well as fluorescence ‘turn on’ probe for the rapid detection of hydrazine

Abstract

A fluorescein based reactive probe has been designed and synthesized to detect hydrazine selectively over other common analytes. We used here 4-bromobutyrate as a masking unit of fluorescein dye. Hydrazine plays here the role of a de-masking agent to set free the fluorescein dye through a simultaneous substitution–cyclisation–elimination process. This leads to ‘turn on’ fluorescence with easily discernible color change with a fast response time (<15 minutes).

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In recent decades, the fluorescence spectroscopic method has been used widely to detect various analytes because of its high sensitivity, specificity, ease of operation, low cost and ability to provide real-time monitoring.¹ Hydrazine is a well known inorganic compound. It is a strong base with good reducing property² and is used in many chemical and pharmaceutical industries as a corrosion inhibitor, catalyst, pharmaceutical intermediate and textile dye etc.³ It is a familiar high-energy fuel and is widely used in rocket-propulsion systems due to its highly flammable nature.⁴ Despite its usefulness, hydrazine has high toxicity and it was also reported that the exposure of hydrazine causes critical damage to kidney, liver, lungs and the nervous system of humans and animals.⁵ Thus, the U.S. Environmental Protection Agency (EPA) classified hydrazine as a probable carcinogen and recommended its threshold limit value (TLV) as low as 10 ppb.⁶

Therefore, the construction of a small molecule fluorescent probe, which can identify trace amounts of hydrazine selectively and delicately, has gained attention in recent years. However, there are only a few hydrazine selective fluorescence chemosensors reported.⁷ There are many chemodosimeters reported based on the deprotection or chemical transformation of a protecting group by the specific deprotecting agent or analyte.⁸ These chemodosimetric systems gained much more attention recently due to their high selectivity. Previously, we have reported a benzothiazole based chemodosimetric system for the selective fluorescence ratiometric detection of hydrazine.⁷ⁱ Although our previous probe showed ratiometric fluorescence change upon reaction with hydrazine, a visible color change, detectable by the naked eye, was not observed. The optical/colorimetric sensors have great demand due to their simple and inexpensive detection methods, i.e., they can be easily observable by the naked eye without using any equipment. In this regard, we choose here the fluorescein dye as a signalling unit due to its capability of both colorimetric and fluorometric signalling, high quantum yield and water solubility.⁹

Based on these considerations, here we would like to report a colorimetric and fluorimetric reactive probe that enables the selective detection of hydrazine through a substitution–cyclisation–elimination pathway. Probe FLB (FL—fluorescein and B—bromobutyric acid) was constructed by connecting 4-bromobutyric acid with fluorescein dye through esterification (Scheme 1). The probe was fully characterized by ¹H NMR, ¹³C NMR and ESI MS spectroscopy (ESI, Fig. S19–S22†).

Scheme 1 Synthetic scheme of the probe (FLB).

As expected, after esterification the dye loses its original fluorescence property and turns colorless. Addition of hydrazine reserved the original fluorescence and greenish-yellow color of the dye through de-masking of the masking group to free the dye itself via a substantial substitution–cyclisation–elimination sequence.

The specific reactivity and sensitivity of the probe (FLB) towards hydrazine was investigated by monitoring its ground state and excited state spectral changes upon addition of different cations, anions, and neutral bases [Cd²⁺, Ag⁺, Pd²⁺, Ni²⁺, Zn²⁺, Cu²⁺, Mn²⁺, Mg²⁺, Fe³⁺, Co²⁺, Cr³⁺, Hg²⁺ (as their chloride salts); F⁻, NO₃⁻, OCl⁻, HSO₄⁻, HSO₃⁻, SO₃²⁻, SO₄²⁻ (as their sodium salts); NH₂(CH₂)₂NH₂, NH₃ and NH₂OH].

Only N₂H₄ has been shown to perturb the photophysical behaviour of FLB with a prominent color change from colorless to greenish-yellow. Other relevant analytes did not affect the absorption profile, they are almost nonresponsive. FLB (20 μM) in CH₃OH–H₂O (1 : 1, v/v, pH = 7.1) solution showed an absorbance maxima at 270 nm corresponding to a closed lactone conformation of fluorescein dye and exhibits no absorption features in the region of 450–550 nm. Upon gradual addition of N₂H₄ to the FLB (20 μM) solution a remarkable increase in the absorbance intensity at 496 nm was observed (Fig. 1). The intensity of the band enhanced regularly with incremental addition of N₂H₄ (0 to 3 equiv.). Further addition of hydrazine lead to a minor change in the absorbance profile of FLB. Accordingly an enhancement (∼27 folds) in the absorbance intensity was observed, accompanied by a prominent colour change from colourless to greenish-yellow. A linear relationship was observed between the absorbance intensity and concentration of hydrazine added in the range of 5–40 μM (Fig. 1, inset).

Fig. 1 Change of absorption spectra of FLB (20 μM) upon gradual addition of hydrazine (0 to 3 equivalents). Insets: the response curve of absorbance of FLB at 496 nm depending on the hydrazine concentration; a photograph of FLB (20 μM), showing visible color change of FLB in the absence and presence of 4 equivalents of hydrazine (right).

In the emission spectra, FLB itself shows very weak fluorescence with an emission maxima centred at 516 nm upon excitation at 450 nm in aqueous methanol solution (CH₃OH–H₂O, 1 : 1, v/v, 0.1 mM HEPES buffer solution, pH = 7.1). To demonstrate the capability of FLB in the determination of N₂H₄ concentration, the probe FLB (20 μM) was treated with various concentrations of hydrazine (0–4 equiv.) solution. Upon interaction with hydrazine, intensity of the emission band (516 nm) increases rapidly with increasing concentration of hydrazine (Fig. 2). There was almost 95 times enhancement of fluorescence intensity observed after addition of 4 equiv. of hydrazine than the FLB itself. The fluorescence quantum yields (Φ) of FLB (20 μM) before and after addition of hydrazine (4 equivalents) were calculated using anthracene as the reference and these were found to be 0.01 and 0.54, respectively (ESI†). Consequently, after addition of hydrazine to the solution of FLB a strong green color fluorescence was observed under the UV light (Fig. 2, inset). This may be due to the opening of spiro-lactone framework of fluorescein dye, which was formed in situ after the reaction of hydrazine with FLB. An excellent linear correlation between the added N₂H₄ concentrations and the fluorescence intensity was observed in the spectra in the range of 2–39 μM with a good R² value of 0.9960 (ESI, Fig. S7†).

Fig. 2 Change of emission spectra of FLB (20 μM) upon gradual addition of hydrazine (0 to 3 equivalents). Inset: visible emission observed from FLB (20 μM) in the absence and presence of 4 equivalents of hydrazine after irradiation under UV light, λ_ex = 460 nm.

The detection limit of the probe for hydrazine was determined from the emission spectral change, upon addition of hydrazine to be 3.881 × 10⁻⁸ M, using the equation¹⁰ DL = K × Sb1/S, where K = 3, Sb1 is the standard deviation of the blank solution and S is the slope of the calibration curve (ESI, Fig. S7†). These results demonstrate that the chemodosimeter (FLB) could detect N₂H₄ quantitatively by the fluorescence spectroscopy method.

To investigate the selective reactivity of the probe (FLB) with hydrazine, absorption and emission titration experiments of the probe in the presence of other analytes were performed. As shown in Fig. 3, the probe displays a selective response towards hydrazine, whereas the other analytes show an insignificant effect on both the absorption and emission spectra of the FLB.

Fig. 3 A comparative view of absorption (a), and emission (b), intensities of FLB (20 μM) upon addition of different analytes (4 equivalents).

In order to investigate the effect of acid and base toward the probe, the change of fluorescence intensity of FLB (20 μM) was measured at different pH values. For this purpose, we have used FLB in a mixture of CH₃OH–H₂O (1 : 1, v/v, 25 °C) solution. From the pH titration experiment it was clear that the chemodosimeter (FLB) showed insignificant change in the pH range of 2–8, indicating that FLB is stable in this pH range and its response toward hydrazine was also almost invariable in this pH range (ESI, Fig. S8†). From this experiment it was clear that the detection of N₂H₄ by this probe (FLB) was not at all hampered in this pH range. Thus we employed the near neutral pH (pH = 7.1) for the detection of hydrazine.

The interference and selectivity are the two very vital parameters of a probe. The interference of other analytes toward the detection of hydrazine was investigated (ESI, Fig. S12†). The UV-vis absorbance and emission intensity of FLB was measured after treatment of 2.0 equivalents of N₂H₄ in the presence of other analytes (3.0 equivalents). The results show that detection of N₂H₄ in the presence of other relevant analytes is not hampered, that is, the interference for the detection of the N₂H₄ was not observed. The selectivity of the probe towards hydrazine is well executed in this section. So that FLB could be used as a selective and sensitive colorimetric and fluorogenic sensor for N₂H₄.

The visible color change (colorless to yellow) and strong green fluorescence after addition of hydrazine, explain the opening of spirolactone framework of fluorescein moiety, which is actually an N₂H₄-induced reaction to set free the fluorescein dye. The reaction of FLB with N₂H₄ is illustrated by involving two steps. As shown in Scheme 2, first, nucleophilic substitution of hydrazine occurs at the bromo group of FLB to generate a free hydrazide group and then in a second step a nucleophilic addition at the carbonyl group from the primary amine group takes place with intramolecular cyclization to release the fluorescein moiety, which turned into its ring open form and is responsible for the color change as well as fluorescence ‘on’.

Scheme 2 Possible mechanism of sensing hydrazine by FLB.

The charge surface diagram of FLB is in favour of the proposed reaction sequence (ESI, Fig. S14†). From the diagram it was clear that the adjacent C-atoms of bromo groups are positively charged (maybe because of the –I effect of –Br group). So, the nucleophilic attack of hydrazine possibly takes place at these centres first. The Gibbs free energy change has been found to be −20 kcal mol⁻¹ of this reaction which is a responsible factor for the conversion of FLB to fluorescein on addition of N₂H₄.

The transformation of FLB to fluorescein after reaction with hydrazine was evidenced by ¹H NMR, ESI MS, UV-vis and emission spectroscopic studies. The reaction product of FLB and N₂H₄ was subjected to mass-spectral analysis and it was found to be (m/z 332.0657) corresponding to fluorescein (ESI, Fig. S23†). The ¹H NMR spectrum of FLB after addition of 5 equivalents of hydrazine looks similar to that of fluorescein in the presence of the same amount of hydrazine (ESI, Fig. S24†). The comparison of UV-vis and fluorescence spectra of FLB and fluorescein after addition of 5 equivalents of hydrazine also supports this fact (ESI, Fig. S10–S11†).

In chemodosimetric systems reaction time, i.e., the response time, is an important factor. Thus we investigated the required reaction time of FLB with hydrazine in 50% methanolic aqueous solution. We recorded the absorption and emission spectral changes of FLB after addition of 5 equivalents of hydrazine in different time intervals (ESI, Fig. S1 and S3†). With increasing time the absorbance at 495 nm and fluorescence intensity at 516 nm increases repeatedly up to about 12 minutes and then reaches a plateau (ESI, Fig. S2 and S4†). From these results, it was concluded that the probe is suitable for the rapid detection of hydrazine.

In order to understand the relationship between the structural changes of FLB to fluorescein on addition of N₂H₄ and their electronic spectra, density functional theory (DFT) and time dependent density functional theory (TDDFT) calculations with the B3LYP/6-31+G method basis set using the Gaussian 03W, Revision-D.01 (ref. 11) program were carried out and visualised using Gauss view program. The optimized geometry and the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of FLB and fluorescein are shown in Fig. 4.

Fig. 4 HOMO and LUMO of FLB and the reaction product of FLB + N₂H₄ (fluorescein) obtained from optimized geometry.

From gas phase TDDFT calculations, a transition at 264.5 nm was calculated which is due to a HOMO to LUMO transition of FLB molecule and this is close to the experimentally observed absorption of 270 nm (ESI, Fig. S15†). In the case of fluorescein, the HOMO to LUMO absorption at 475.1 nm compliments the greenish yellow colour of the solution (ESI, Fig. S16†). When solvent correction was incorporated by CPCM model¹² and 1 : 1 mixture of water and methanol were chosen as solvent the calculated absorption spectra are observed at 268.3 nm and 481.2 nm for FLB and fluorescein, respectively, which is due to the HOMO to LUMO transition (ESI, Fig. S17–S18†).

Here we also explored the practical applications of FLB toward the detection of hydrazine, because of its carcinogenic property and huge use in a variety of industrial processes. The chemodosimetric attacking of hydrazine towards the probe gives us an opportunity for the detection of hydrazine in aqueous samples. Both tap water and distilled water have been analysed to examine the “tap-water application”. An aliquot of hydrazine was added to both of the tap and distilled water and the recovery of hydrazine by FLB was examined through fluorescence study. Now the healing of hydrazine by FLB was investigated from these two water samples and the examination of these two solutions explored that hydrazine in both solutions established well up to 40 μM concentrations (Fig. 5).

Fig. 5 Fluorescence detection of hydrazine in distilled water and tap water by FLB. [FLB] = 10 μM, [hydrazine] = from 0 to 40 μM, in a mixture of CH₃OH and HEPES buffer solution (pH = 7.1) (1 : 1, v/v), λ_ex = 450 nm.

In summary, we present here the design and synthesis of a reactive probe using fluorescein dye, for the selective detection of hydrazine. The probe (FLB) shows significant enhancement in both emission and absorption spectra upon interaction with hydrazine due to the opening of spirolactone framework of fluorescein dye. The reaction of hydrazine with the probe (FLB) to free the fluorescein dye goes through the substantial substitution–cyclization–elimination sequence. The sensing mechanism was supported by ¹H NMR, ESI MS and TDDFT studies.

Acknowledgements

Authors thank DST and CSIR, Govt of India, for financial support. S. D., K. A., and B. P. acknowledge CSIR for providing them with fellowships.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental procedure, synthesis of the probe, NMR, MS, computational data etc. See DOI: 10.1039/c3ra46663a

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