Rajkishor Majia,
Ajit Kumar Mahapatra*a,
Kalipada Maitia,
Sanchita Mondala,
Syed Samim Alia,
Prithidipa Sahoob,
Sukhendu Mandalc,
Md Raihan Uddinc,
Shyamaprosad Goswamia,
Ching Kheng Quahd and
Hoong-Kun Funde
aDepartment of Chemistry, Indian Institute of Engineering Science and Technology, Shibpur, Howrah-711103, West Bengal, India. E-mail: mahapatra574@gmail.com; Fax: +91 3326684564
bDepartment of Chemistry, Visva-Bharati (A Central University), Santiniketan 731235, India
cDepartment of Microbiology, University of Calcutta, Kolkata-700019, India
dX-ray Crystallography Unit, School of Physics, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia
eDepartment of Pharmaceutical Chemistry College of Pharmacy, King Saud University, P.O. Box. 2457, Riyadh 11451, Kingdom of Saudi Arabia
First published on 15th July 2016
A new probe 2-benzo[1,2,5]thiadiazol-4-yl-isoindole-1,3-dione (BTI) based on the Gabriel reaction mechanism was synthesized and characterized for the specific detection of hydrazine with high selectivity against other amines in an organo-aqueous solution. Upon hydrazinolysis of BTI in the presence of hydrazine in a H2O–DMSO (4:
6, v/v) solution (10 mM HEPES buffer, pH 7.4) at room temperature, the chemosensor produces fluorescent aminobenzthiadiazole with a maximum emission at 498 nm along with a color change from colorless to green, allowing selective colorimetric and fluorometric detection of hydrazine by the naked eye. Probe BTI was also successfully applied in vapor phase hydrazine detection into a solid state over other interfering volatile analytes. Furthermore, the probe BTI coated with silica gel TLC plates could act as a visual and fluorimetric probe for hydrazine vapor detection. The experimental detection limit of hydrazine is 2.9 ppb, which is well below the accepted limit (10 ppb) for hydrazine set by the U.S. Environmental Protection Agency (EPA). DFT and TDDFT calculations were performed in order to demonstrate the sensing mechanism and the electronic properties of probe and hydrazinolysis products. Additionally, probe BTI could also be applied for the imaging of hydrazine in living cells.
Various traditional analytical techniques for the detection of hydrazine are available, including electrochemical analysis9 and chromatography,10 including gas chromatography,11 HPLC,12 coulometry,13 potentiometry,14 titrimetry,15 capillary electrophoresis16 and electroanalysis.17 Spectrophotometry using colored derivatives, such as p-dimethylaminobenzaldehyde18 and chlorosalicylaldehyde,19 are also used to detect hydrazine. However, these methods are not only complex and time consuming, but also impractical for in vivo hydrazine analysis because of their post-mortem processing and destruction of tissues and cell contents. Among several detection strategies, fluorescent techniques are extremely attractive due to their high sensitivity, low cost, easy implementation, and real-time detection.20,21 Till now, only a few fluorescent chemodosimeters for hydrazine have been reported, and almost all of them were designed based on the deprotection or chemical transformation of a protecting group by a specific deprotecting agent or analyte.22–26 For example, the fluorescent sensing system developed by Chang et al.,27 showed the selective deprotection of levulinated coumarin in presence of hydrazine in DMSO–water. The sensing system reported by Peng et al.,28 showed a ratiometric hydrazine-selective NIR probe based on cyanine dye via deprotection of acetyl group in aqueous-organic solvent. Therefore, it still remains a challenge to develop effective hybrid fluorescent probes with suitable reactive zone that can act as good chemodosimeter for the recognition of molecular species, though such systems are limited in literature in case of hydrazine sensing. Furthermore, fewer sensors have been applied to vapor sensing.29,30 Cui et al.29 have reported an efficient fluorescent chemodosimeter which employs a naphthalic anhydride fluorophore. It senses hydrazine in an elegant manner by the Gabriel reaction. Keeping this in mind, we have envisioned a chemodosimeter which contains benzothiadiazole fluorophore which incorporates excellent turn-on fluorescence properties along with enhanced bio-compatibility. Some of the current probes could only be utilized at low pH (pH < 5) conditions31,32 which would limit their application in physiological conditions. Thus, developing a new fluorescence method of monitoring hydrazine in living cells or sensing vapor phase hydrazine remains a significant challenge.
In continuation of our research work in the development of various fluorescent chemosensors for important toxic analytes, herein, we disclose the design and synthesis of a fluorescence sensor based on phthalimide–benzothiadiazole molecular hybrid, which can selectively detect hydrazine in aqueous-DMSO media. The selection of the hybrid phthalimide–benzothiadiazole platform is due to favorable photophysical properties of aminobenzothiadiazole including high quantum yields, high extinction coefficient, and emission maximum beyond 450 nm in the visible region. We recently reported the selective detection of biothiols and hydrazine using benzothiadiazole, BODIPY–pyrene and carbazole based chemosensor.33–35 Therefore, it is of prime interest to develop phthalimide–benzothiadiazole-based hybrid reactive molecular systems to provide better sensitivity and selectivity toward sensing of toxic molecules. However, a benzothiadiazole-based fluorescent probe for hydrazine detection has not been widely reported. To our knowledge, there are very few reports36–38 of selective hydrazine fluorescent probes based on Gabriel phthalimide type moiety. It is important to note that our new probe, 2-benzo[1,2,5]thiadiazol-4-yl-isoindole-1,3-dione (BTI) is highly sensitive to hydrazine with detection limit of 8.47 × 10−8 M.
Fluorescent hydrazine probe has been constructed by exploiting the high nucleophilic reactivity of the hydrazine molecule. In this work, we judiciously designed probe BTI (Scheme 1) as a new type of selective hydrazine fluorescent probe based on the Gabriel type hydrazinolysis of benzothiadiazole derivative of phthalimide. Probe BTI contains a aminobenzothiadiazole moiety which acts as fluorescent signal transducer and a phthalimide moiety for reacting zone. It is known that in Gabriel method N-substituted phthalimide reacts with hydrazine via simultaneous substitution–elimination process twice gives phthalhydrazide and free primary amine. Due to a photoinduced electron transfer (PET) process from the electron donor, fluorophore to the electron accepter phthalimide(isoindole-1,3-dione) moiety, probe BTI is non-fluorescent (Φ = 0.067). However, when treated with hydrazine, it exhibits a relatively rapid, time-dependent enhancement of its fluorescence signal (Φ = 0.704). Such finding suggests that hydrazine selectively reacts with phthalimide moiety in probe BTI, thus eliminating PET-induced fluorescence quenching and we should observe a substantial fluorescence turn-on response due to free aminobenzothiadiazole moiety.
Φx = Φs(Fx/Fs)(As/Ax)(nx2/ns2) |
Indeed, as designed, probe BTI is essentially weak-fluorescent (Φ = 0.067) in the neutral aqueous conditions H2O–DMSO (4:
6, v/v) solution (10 mM HEPES buffer, pH 7.4), whereas aminobenzothiadiazole 2 is highly fluorescent (Φ = 0.704) around 498 nm in the same aqueous buffer. Thus, it is apparent that compound BTI is promising as a fluorescence turn-on probe for hydrazine provided that compound BTI could be converted by hydrazine to give fluorescent free amine 2.
A summary of the crystallographic data is given in Table S1 in the ESI.† The BTI molecule, Fig. 1a, is twisted with a dihedral angle between the benzothiadiazole (S1/N2/N3/C10–C14, r.m.s. deviation = 0.009 Å) and isoindole ring systems (N1/C1–C18, r.m.s. deviation = 0.022 Å) is 51.61(5)°. In the crystal (Fig. 1b), the molecules are linked by intermolecular C–H⋯O and C–H⋯N hydrogen bonds (Table S2 in the ESI†) and resulting in two-dimensional planes parallel to (011) (Fig. 1b). The molecular structure is further stabilized by weak aromatic π–π stacking interactions between the benzene and thiadiazole rings of adjacent molecules [centroid–centroid separation = 3.7002(10) Å].
Refinement parameters of BTI (Cambridge Crystallographic Data Centre as entry CCDC 1479814†):
The sensitivity of BTI toward different amines and their preferential selectivity toward hydrazine over the other amines has been studied by absorption and fluorescence titrations. We found that the addition of trace amounts of hydrazine causes the absorption and fluorescence signal to change rapidly, which is very important for real-time detection. Therefore, the following titration experiments were carried out after adding varying concentrations of hydrazine (Fig. 2) to a fixed concentration of BTI in H2O–DMSO (4:
6, v/v) solution (10 mM HEPES buffer, pH 7.4).
In a UV-vis absorption spectrum, the solution of BTI showed dual absorption bands at 306 and 313 nm which may be attributed to π–π* as well as vibronic transitions. Upon the addition of hydrazine, the dual absorption bands gradual decreases with a concomitant growth of a new structureless band at 395 nm, therefore induce a color change from colorless to yellow (Fig. 2). The presence of clear isosbestic point at 362 nm implies that it transforms quantitatively to a new species.
The probe BTI exhibit very weak emission at ∼498 nm when excited at 313 nm in H2O–DMSO (4:
6, v/v) solution (10 mM HEPES buffer, pH 7.4).
Addition of increasing concentrations of hydrazine to the probe BTI results in the enhancement of fluorescence intensity at 498 nm as a function of the added hydrazine concentration, and the fluorescence enhancement at 498 nm was up to 9.05-fold (Fig. 3). Furthermore, the introduction of hydrazine turned the visual emission of the probe BTI solution from dark to bright green (Fig. 3a, inset), which further supports the fluorescence turn-on response.
The changes in the fluorescence spectrum stopped when the amount of added hydrazine reached 1.5 equivalent of the probe. A plot of fluorescence intensity as a function of added [hydrazine]/[BTI] mole ratio (Fig. 3b, inset) shows a stoichiometry of 1:
1 between the probe and hydrazine and the intensity goes to highest value at ≥1 equiv. A linear relationship was observed between the fluorescence intensity and hydrazine amount in the range of 0.025–1.5 μM. The detection limit of probe BTI towards hydrazine was calculated to be 8.47 × 10−8 M (2.9 ppb) (Fig. S5 in the ESI†) which was lower enough than that of the TLV (10 ppb) recommended by the EPA and WHO.
To confirm that the fluorescence sensing response of the probe to hydrazine is indeed due to the conversion of probe BTI to compound 2, the reaction product of probe BTI with hydrazine was isolated by column chromatography. The 1H NMR spectrum of the isolated product is essentially identical with that of the standard compound 2 (Fig. S6 in the ESI†), in good agreement with the formation of compound 2. On the basis of these experiments and reported literatures,36–38 we speculate that the carbonyl position of phthalimide in the BTI was selected as the reaction site and the proposed reaction mechanism of BTI with hydrazine is illustrated in Scheme 2 involving two steps. At first the nucleophilic addition–elimination to the carbonyl group at the phthalimide in the BTI resulted in the intermediate I and then the second nucleophilic addition–elimination to the another carbonyl group by –NH2 in I resulted amide ring formation that leads to phthalhydrazide and release the aminobenzthiadiazole (2), which carry out a unique chromogenic response. To confirm the validity of the proposed sensing mechanism, a solution of probe BTI was analyzed by 1H NMR in the absence and presence of hydrazine, as displayed in Fig. 4.
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Fig. 4 Partial 1H NMR (400 MHz) spectra of BTI only and [BTI + N2H4]. [BTI] = 1 × 10−3 M, [hydrazine] = 1 × 10−2 M in D2O/DMSO-d6 (4![]() ![]() |
After hydrazine was added, the protons Ha and Hc of probe BTI moved up field from 8.305, 7.939 to 6.597 and 7.143 ppm respectively, which was almost identical to that of aminobenzthiadiazole (2) (Fig. S6 in the ESI†). A new peak at 6.164 ppm also appeared, assignable to the corresponding –NH2 protons (Hg) of 2. In addition, the characteristic NH proton resonances of the reaction product phthalhydrazide was also clearly observed at 8.028 ppm (Fig. 4) indicating the hydrazinolysis of phthalimide moiety. To further understand the mechanism of probe BTI with hydrazine, LCMS was used to test the solutions containing BTI and 2 equiv. hydrazine, and the peak at 163.2 instead of 282.1 proved that the benzthiadiazole group had been removed and generation of new peak at 169.3 corresponds to aminobenzthiadiazole (2) (Fig. S7 in the ESI†). Thus, the extensive studies of NMR, mass spectrometry, absorption, emission, and excitation spectroscopy corroborate that indeed, as designed, nonfluorescent probe BTI was transformed by hydrazine to afford strongly fluorescent compound 2 for a fluorescence turn-on response (Scheme 2).
In order to check the practical utility of BTI to detect hydrazine selectively even in the presence of common anions and cations, redox molecules and amines competitive analyte titrations were carried out. The BTI fluorescent probe displayed a large fluorescence turn-on response to hydrazine (Fig. 3). By contrast, representative species such as F−, Cl−, Br−, I−, CN−, PO43−, NO3−, S2− and SO42− (as their sodium salts), and amines (ethylenediamine, 1,3-diaminopropane, o-phenylenediamine, ammonia, hydroxylamine, cysteine, homocysteine, urea and thiourea) exhibited almost no changes in emission behaviour. Meanwhile, the commonly encountered cations [Cu2+, Hg2+, Zn2+, Cd2+, Mg2+, Fe3+, Al3+ and Ag+ (as their chloride salts)] did not cause any change to the fluorescence of probe BTI (Fig. S8 in the ESI†).
However the absorption and fluorescence titration carried out with all the other common cations and anions as well as redox anions showed no significant change, indicating their noninteractive nature with BTI (Fig. 2b and 5a). Even the different amines do not react to BTI at room temperature (Fig. 3b and 5b). The results indicate that the probe BTI has high selectivity for hydrazine over other species. This may be attributed to the unique chemical reaction between phthalimide and hydrazine. Fluorescence spectra were also recorded for the titration of probe BTI against hydrazine in the presence of 50 equiv. of common anions and cations, redox anions and amines. None of these analyte significantly affect the emission intensity of BTI upon the addition of hydrazine, and the titration profile is similar to that obtained for simple hydrazine titration (Fig. 3b and 5a). Therefore, it can be concluded that probe BTI selectively reacts hydrazine even in the presence of other analytes.
The time course of the fluorescence intensity of the probe BTI (c = 1 × 10−6 mL−1) in the absence or presence of hydrazine (1.5 equiv.) in H2O–DMSO (4:
6, v/v) solution (10 mM HEPES buffer, pH 7.4) is displayed in Fig. S9 in the ESI.† The free probe BTI exhibited no noticeable changes in the emission intensity at 498 nm. However, upon introduction of hydrazine, a significant enhancement in the emission intensity was observed within minutes, and the emission intensity essentially reached the maximum in 20 minutes. To be useful in biological applications, it is necessary for a probe to function over a suitable range of pH, in particular at physiological pH. So, the effect of pH on the fluorescence response of BTI to hydrazine was investigated. As shown in Fig. S10 in the ESI† in the absence of hydrazine, almost no change in fluorescence intensity was observed in the free chemosensor over a wide pH range of 1.0–11.0, indicating that the free chemosensor was stable in the wide pH range. Therefore, considering the environmental and biological applications, all studies were carried out at the physiologically relevant pH of 7.4 for the detection of hydrazine.
To get insight into the optical response of probe BTI to hydrazine, probe BTI and the corresponding product after reaction with hydrazine 2 and 3 were examined by density function theory (DFT) and time-dependent density function theory (TDDFT) calculations using a TDDFT//B3LYP/6-31+G(d,p) + solv (SMD) level of the Gaussian 09 program.40 Geometries have been optimized in presence of solvent water. Solvent effects were incorporated using SMD solvent model. The optimized geometries and calculated electron distributions in the frontier molecular orbitals of BTI, 2 and 3 are shown in Fig. S12 in the ESI.†
In addition, we also performed time-dependent density function theory (TDDFT) calculations for the reactant as well as both the product also. The vertical transitions i.e., the calculated λmax, main orbital transition, and oscillator strength (f) are listed in Tables S3 and S4 (ESI†). In the case of the BTI probe, TDDFT calculations provided absorption band at ∼336 nm belonging to the S0 → S2 (f = 0.3052) energy state. This value is consistent with the absorbance band at 313 nm (ε = 3.78 × 105 M−1 cm−1) obtained experimentally. Furthermore, the energy gap between the HOMO and LUMO of 2 was smaller than that of probe BTI, in good agreement with the apparent red shift (∼395 nm) in the absorption observed upon the treatment of probe BTI with hydrazine. The calculated band at ∼466 nm of 2 is assigned to the vertical major transition of HOMO → LUMO, S0 → S1 (∼98.80%) that results from an n → π* transition within the amino-benzothiadiazole moiety of 2, which mainly corresponds to the experimentally observed absorbance band at 395 nm (Table 1).
Molecules | Electronic transition | Experimentally obtained (λmax) | Theoretically obtained (λmax) | fb |
---|---|---|---|---|
BTI | S0 → S2 | 313 nm | 336.87 nm | 0.3052 |
2 | S0 → S1 | 395 nm | 466.37 nm | 0.0571 |
3 | S0 → S1 | 315 nm | 290.97 nm | 0.0647 |
To be useful in practical applications, we further tested whether probe BTI could be applied for the detection of gas state hydrazine. To make the detection experiments easy to perform and practical, silica gel TLC plates were used. Prior to detection, silica gel TLC plates (silica layer of thickness 0.2 mm on aluminium foil) were prepared by immersing the TLC plates into a CHCl3 solution of probe BTI (c = 1 × 10−3 mL−1) and then dried. The probe-loaded TLC plates were covered on the top of jars that contained different hydrazine solution concentrations (blank, 0.01%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 25%, 30% and 40% in water) for 15 min at room temperature before it was ready to observe. As shown in Fig. 6, the change in the color of the fluorescence from colorless to green was observed using a hand-held UV lamp with excitation at 366 nm.
The gas state hydrazine detection limit of probe BTI concentration is as low as 0.1%, which is considerably more sensitive than the recently developed hydrazine probes.29,30 It is noteworthy that the probe has high potential applications in hydrazine detection. Hydrazine in gaseous form often threatens human life, so our designed probe has more application potential.
We further tested whether probe BTI could be applied for the detection of hydrazine in solution. To make the detection experiments easy to perform and practical a TLC plate was used. Prior to detection, a silica gel TLC plate (silica layer of thickness 0.2 mm on aluminium foil) was firstly immersed into H2O–DMSO (4:
6, v/v) solution (10 mM HEPES buffer, pH 7.4) solution of BTI (c = 1 × 10−3 mL−1) and dried, then the probe-loaded TLC plate was sink into a beaker containing hydrazine solution for 1.0 min at r.t. before it was ready to observe. As shown in Fig. 7, the change in the color of the fluorescence from dark to green was observed using a hand-held UV lamp with excitation at 366 nm.
We also explored opportunities for probe BTI to analyze hydrazine in aqueous solution for practical applications. Because hydrazine has carcinogenic properties, and has been widely used in a variety of industrial processes, hydrazine detection in aqueous samples is of interest. Prior to living cell imaging, probe BTI was used to detect hydrazine in tap water and distilled water. An aliquot of hydrazine was added to water and the recoveries obtained by BTI signals were compared in tap water and distilled water (Fig. 8). The analysis of hydrazine in both solutions agreed well at hydrazine concentrations up to 10 μM. The results show that probe BTI can detect hydrazine in real water samples quantitatively.
To further demonstrate the application potential of probe BTI in living cells, the probe was applied in Vero cells for fluorescence imaging of hydrazine. Prior to investigating the suitability of the probe BTI for imaging hydrazine in living cells, it is necessary to evaluate its cytotoxicity. The standard MTT assays suggest that the probe BTI does not exert any adverse effect on cell viability (Fig. S13 in the ESI†). Now the stage was set for cell imaging of hydrazine. The living cells were treated with the probe BTI in the absence or presence of hydrazine. Vero cells incubated with BTI (10 μM) for 20 min at 37 °C in PBS buffer with 0.5% DMSO showed nonfluorescent as shown in Fig. 9. By contrast, cells pre-loaded with the probe BTI and further incubated with hydrazine for further 10 min displayed green fluorescence (Fig. 9) inside Vero cells, as observed earlier in solution studies. These findings open up the avenue for future in vivo biomedical applications of the sensor. Thus, BTI is cell membrane permeable and capable of fluorescence imaging of hydrazine in the living cells.
Footnote |
† Electronic supplementary information (ESI) available. CCDC 1479814. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra14212e |
This journal is © The Royal Society of Chemistry 2016 |