Results and Discussion

Synthesis and Characterization of Receptor 1

The fluorescent receptor 1 was concisely synthesized from 2-thiophenecarboxaldehyde and 1-acetylpyrene in a single step condensation reaction (Scheme 1). The final receptor 1 was confirmed by NMR (1H and 13C) and mass spectrometry. (Supp. Info. S1-S3). The chemosensor is designed in such way that, it has a receptor-linkerfluorophore unit. The pyrene scaffold acts as a fluorophore, which is covalently attached to a thiophene moiety (receptor) through a C=Cspacer unit. The hetero atoms, oxygen and sulphur act as binding sitesfor the metal ion detection.

Selectivity studies of receptor 1

The fluorescence response of the chemosensor 1 towards various metal ions were performed at a physiological pH in DMSO-H2O (1:1 v/v, HEPES=50 mM, pH=7.4) solution. Upon addition of 100 equiv. of Pb²⁺ to a solution of 1, significant enhancement of fluorescence with an emission maximum at 418 nm was observed compared to the sensor alone under the same experimental conditions. However, under the identical concentration of other metal ions (Na⁺, K⁺, Al³⁺, Cu²⁺, Cd²⁺, Hg²⁺, La²⁺, Zn²⁺, Co²⁺, Ni²⁺, Ca²⁺, Mn²⁺, Cr³⁺, Ba²⁺, Ce³⁺, Mg²⁺, Fe²⁺, Fe³⁺ and Ag⁺) sensor 1, produced no considerable changes in the fluorescence spectra (Figure 1). Therefore, 1 act as a highly selective fluorescence ‘‘off-on’’ probe for Pb²⁺ in DMSO-H₂O (1:1 v/v, HEPES=50 mM, pH=7.4) solution.

For practical applications, the possible interferences by other metal ions were carried out through competitive experiments. The fluorescence changes of 1 was measured by the treatment of 100 equiv. Pb²⁺ ions in the presence of same equiv. other interfering metal ions including Na⁺, K⁺, Al³⁺, Cu²⁺, Cd²⁺, Hg²⁺, La²⁺, Zn²⁺, Co²⁺, Ni²⁺, Ca²⁺, Mn²⁺, Cr²⁺, Ba²⁺, Ce²⁺, Mg²⁺, Fe²⁺, Fe²⁺ and Ag⁺. The tested interfering metal ions showed no observable interference with the detection of Pb²⁺ ion (Figure 2). These results suggested that receptor 1 could be used for the selective detection of Pb²⁺-ion in environmental and biological sample analysis.

Fluorescence responses and stoichiometric studies

Furthermore, the fluorescence response of 1 to various concentrations of Pb²⁺ was investigated as titration experiments. As shown in Figure 3, upon addition of Pb²⁺ (0-60 equiv.), the fluorescence of 1 gradually increased and remained steady when 100 equiv. of Pb²⁺ was added, implying that 1 interacts with Pb²⁺ in 2:1 stoichiometry. The Job’s plot method (Figure 4) is used to find the complexation mode between receptor 1 and Pb²⁺ (Host-Guest) complex [37]. The complex showed a maximum mole fraction of 1 is 0.3 at 418 nm established a 2:1 (1:Pb²⁺) binding stoichiometry. The detection limit of the sensor 1 is found to be 0.11 × 10^-6 M^-1, which is calculated using 3δ/S [38].

The binding stoichiometry is further confirmed by the Benesi–Hildebrand plot between 1-Pb²⁺ (Figure 5) [39]. The association constant was determined to be Ka = 1.18 x 105 M^-2 for the 1-Pb²⁺ complex.

The recognition reversibility of 1 to Pb²⁺ was verified with the help of EDTA. As already discussed, sensor 1 alone show a weak fluorescence and after addition of Pb²⁺ there is a significant enhancement of the fluorescence intensity as an turn off-on process. Nevertheless, upon addition of EDTA to the same solution thefluorescence intensity got reduced and peak overlapped with that of the receptor 1 as an turn on-off process. This study indicates that the Pb²⁺ recognition process is reversible one as shown in Figure 6.

The IR spectrum of chemosensor 1 exhibited characteristic IR band in the region of 1429 and 1668 cm^-1 corresponding to C=C and C=O stretching groups. However, as shown in Figure 7, in the IR spectra of 1+Pb²⁺, C=C stretching band is shifted to 1435 cm^-1 and at the same time the C=O group shifted to 1660 cm^-1 [40,41]. These differences in the IR band shift further corroborates that the carbonyl group took part in coordination with Pb²⁺ ions.

Proposed coordination mode of the complex

The proposed coordination mechanism of 1-Pb²⁺ was investigated through fluorescence changes as shown in Scheme 2. For the sensor 1, have no accountable fluorescence (turn-off) due to intramolecular photo-induced-electron-transfer (PET) process within the chalcone based pyrene unit [42-44]. Upon the addition of Pb²⁺-ions to 1, there is a gradual increase in the fluorescence (turn-on) enhancement which eventually inhibits the PET process. By combing all the above results such as, fluorescence titration spectrum, job’s plot and non linear curve fitting methods the possible coordination modes of 1-Pb²⁺-ion complex was found to be 2:1 (Host: Guest) binding stoichiometry.

In addition, a pH titration experiment showed that receptor 1 and 1+Pb²⁺ had stable fluorescence properties over a wide pH range of 5-9 (Figure S4), which suggests that receptor 1 is suitable for biological application under the physiological conditions. The effective time taken by the receptor 1 for the detection of Pb²⁺ was also studied. The bar diagram (Figure S5) reveals that the receptor 1 can complex with 100 equiv. Pb²⁺ completely in 2 min. However, after that no considerable change in the fluorescence intensity was observed.