Design and synthesis of 2-aminothiazolesalicylaldehyde
Receptor1 was synthesized by condensation of 2-Hydroxy benzaldehyde and 2-amino thaizole in methanol with 88% yield as shown in scheme 1 and characterized by 1H-NMR and 13 C-NMR. In receptor1 benzaldehyde group acts as fluorophore, imine group act as spacer, and heterocyclic thaizole group containing N and S atom acts as a binder. The imine group links the fluorophore and binding unit. From FTIR analysis the presence of skeletal stretching of aromatic group is confirmed from the peak at 1600.92 cm-1, the peak at 1192.21 cm-1 showed C-O stretching vibration of alcohol and the peak at 1492.00 cm-1 confirms C-C stretching vibration, the peak at 756.00 cm-1 confirms C-S stretching vibration, the peak at 2742.78 cm-1 confirms C-H stretching vibration. The peak at 1658.78 cm-1 confirms the presence of C=N stretching band (Fig. S1) [31]. The total number of carbon was confirmed by 13C NMR. The chemical shift were noticed at (100 MHz, DMSO: d6, ppm) 130.14, 122.72, 128.32, 117.70, 154.62, 128.53, 140.48, 139.36, 128.96, 129.07.The total number of hydrogen was confirmed by 1H NMR. The chemical shift of H1 NMR (400 MHz, DMSO: d6, ⸹, ppm) (J, Hz): 7.01, 7.12, 7.15 & 7.17 (d, 4H, Ar-H), 6.94 (s, 1H, CH=N), 6.85&6.94 (d, 2H, CH2) and 9.23 (s, 1H, 1-OH) (Fig. S2 & S3) [32]. Analytically Calculated for C10H8N2SO (204.23): C, 120.10; H, 8.06; N, 28.01.
Selectivity Studies
The selectivity studies are performed to assess the sensing behaviour of receptor1 for a selective detection of metal ion. The metal ions such as Ag+, Al3+, Ba2+, Bi3+, Ca2+, Cd2+, Co2+, Cr3+,Cu2+, Fe2+, Fe3+, Hg2+, K+, Li+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Sr2+, Zr2+ and Zn2+ are taken in the form of nitrate salts and added to receptor1 and buffer solution. The excitation wavelength is fixed as 275 nm from UV-Vis spectroscopy. The receptor1 responded to a drastic quenching effect with Fe2+ and Ag+ ions as shown in (Fig.1). However, other metal ions showed no significant changes during the identical condition. As a result, the receptor1 showed a potential response for Fe2+ ion with a turn on-off fluorescence through a possible reverse PET mechanism.
Proposed Binding Mechanism
In PET based sensors, a fluorophore is usually connected via a spacer to a binding unit containing a relatively high energy non-bonding electron pair found in nitrogen donors, capable of transferring an electron to the excited fluorophore to quench its fluorescence (off-on) [33]. The quenching takes place because of the deactivation of the excited state of fluorophore by the addition of electron to its excited state frontier orbitals from the functional group of the fluorophore that quench the fluorescence intramolecularly [34]. This leads to the nonemissive state of fluorophore. Here, in receptor1 the free electron pair of nitrogen present in thaizole and C=N group attached to the fluorophore has strong fluorescence emission, and binding of cation results in weak fluorescence emission because the Fe2+ ion bound to receptor1 is paramagnetic in nature with unfilled d-subshell that strongly quench the fluorescence intensity of the fluorophore through electron transfer between receptor1 and Fe2+ ion [35]. The Fe2+ ion bound to the fluorophore acts as a signal transduction unit and also bound with a thaizole moiety and spacer [36]. There is no possibility of Fe2+ ion to bind with sulphur because of the weak interaction between Fe2+ and sulphur [37]. Therefore, the quenching response of receptor1 towards Fe2+ ascribed to electrons of fluorophore transferring to binding unit by metal ion complexation (reverse-PET process), resulting in fluorescence quenching (Scheme 2) [38].
Interference Study
Interference study was carried out to predict if any competitive metal ion interferes during complexation of receptor1 with Fe2+. Fig.2. shows a change in the fluorescence intensity with respect to the addition of metal ion to receptor1. The emission intensity of receptor1 with various metal ion such as Al3+, Ba2+, Bi3+, Ca2+, Cd2+, Co2+, Cr3+,Cu2+, Fe3+, Hg2+, K+, Li+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Sr2+, Zr2+ and Zn2+ is on par to the emission intensity of receptor1 alone, whereas the emission intensity of receptor1-Ag+ is low compared to the emission intensity of other receptor1-metal ion complex. This is because the electronic configuration of Ag+ ion in 4d subshell is completely filled and results in diamagnetic property, which leads to the decrease in emission intensity as there is no transfer of electron taking place between Ag+ and receptor1. The response of receptor1 specifically towards Fe2+ is based on HSAB (Hard-Soft and Acid-Base) theory, the borderline base (C=N) group is able to coordinate with a borderline acid Fe2+ ion [39, 40]. The receptor1 showed a quenching in fluorescence intensity when it is complexed with Fe2+ is due to the reverse PET mechanism. The tested interfering metal ions showed no obvious interference when receptor1 is complexed with Fe2+ ion (Fig. 2). This result implies that receptor1 could be used for the selective detection of Fe2+ ion in various applications.
Binding and stoichiometry studies
In order to find the binding ability of receptor1 with Fe2+ion, the fluorescence study was carried out by gently increasing the concentration of Fe2+ ion from 0-85 equivalents. A decrease in fluorescence intensity was observed with increase in Fe2+ ion concentration (Fig. 3). The fluorescence intensity was almost saturated upon addition of 85 equivalents of Fe2+. The binding ratio of ligand and complex was predicted by Job’s plot. The Job’s plot confirms the binding mode of the host-guest complex, the maximum mole fraction of complex was 0.5 at excitation wavelength 275 nm (Fig. 4). Job’s plot concludes that the receptor1 interacts with Fe2+ at 1:1 stoichiometry. The stoichiometry binding of receptor and metal was predicted theoretically with the help of Benesi-Hildebrand non-linear fitting curve (Fig. 5) [41-43]. The association constant (Ka) of receptor1-Fe2+ ion is 2.22x102 M-1. The limit of detection (LOD) calculated using 3⸹/S is 33.7x10-9 M, where ⸹ is the standard deviation of the blank signal, and S is the slope of the linear calibration plot indicating correlation coefficient (R2) value of 0.9863 derived from the linear graph plot (Fig. 5).
Reversibility studies
The synthesized chemosensor has a very good reversible property that could be applied for various applications. The reversible property of receptor1 is analysed using a chelating agent EDTA (Ethylenediaminetetraacetic acid). Figure 6a. depicts the reversible property of receptor1. The fluorescence intensity is very high for receptor1. On addition of Fe2+ ion to the receptor1 the fluorescence intensity is quenched due to the complexation of receptor1 with Fe2+ ion. Further on addition of EDTA to receptor1 and Fe2+ ion solution the fluorescence intensity is enhanced. This results shows that receptor1 has a good reversible property. This is confirmed further by the addition of Fe2+ ion and EDTA several times to assess the reproducibility of the reversible property. The change in intensity with respect to Fe2+ and EDTA is due to the complexation of EDTA and Fe2+ ion with receptor1. The cycle was repeated for 10 times (Fig. 6b).
Effect of pH and Time
The pH study is carried out to assess the potential of the synthesized receptor1 to work under various environmental conditions. The effect of pH on the receptor1 and receptor1-Fe2+ complex is done by adjusting the pH from acidic to basic (2 to11) condition. The fluorescence intensity of receptor1-Fe2+ complex decreases in acidic pH and increases at neutral pH and again decreases at basic pH (Fig. 7). The decrease in fluorescence intensity at acidic pH is because the imine (C=N) group of receptor1 gets protonated due to the increase in H+ ion concentration and cleavage may takes place between the fluorophore and heterocyclic group that hinders the binding of Fe2+ ion with receptor1 [44]. On the other hand, even at basic condition the fluorescence intensity decreases. This is because of the deprotonation of H+ ion from phenolic group of fluorophore due to the increase in the concentration of –OH ion. After deprotonation, the Na+ ion occupies the phenoxide group and results in O-Na+ which hinders the binding of Fe2+ ion with receptor1 leading to a decrease in fluorescence intensity. The change in fluorescence intensity at acidic and basic condition is due to the presence of fluorophore group in receptor1. The increased fluorescence intensity at 7.4 indicates that the receptor1 works at physiological pH 7.4 which is suitable for biological and environmental application. Hence all the other experiments are carried out at pH 7.4.
A study was carried out further to assess the time taken for the complex formation between receptor1 and Fe2+ [45].The decrease in fluorescence intensity from 0 to 4th minute showed that the complexation between receptor1 and Fe2+ ion was unstable. After 4th minute the saturation in graph indicates the stable complexation between receptor1 and Fe2+ ion. The solution was kept further for 60 minutes and the fluorescence intensity was noted. There were no desirable changes after four minutes (Fig. 8). Hence, the result shows that the receptor1 favours for detection of Fe2+ ion in short interval of time.
Application studies
Determination of Fe2+ ion in water sample
The synthesized receptor1 was subjected to the detection of Fe2+ ion in water sample. Three different water samples (Bore well water (B1-3 µM, B2-6 µM), Tap water (T1-3 µM, T2-6 µM), and RO drinking water (D1-3 µM, D2-6 µM)) are collected from Coimbatore Institute of Technology. The sample water is spiked with Fe2+ ion concentration of 3 and 6 µM. About 2 mL of 4x10-6 M concentration of receptor1 was added to the spiked water sample. The recovery percentage of Fe2+ ion from spiked water sample after adding receptor1 is calculated from fluorescence spectroscopy (Fig. S4). The receptor1 showed the enhanced fluorescence intensity but on adding the spiked water sample with receptor1 the quenching in fluorescence intensity is noticed. The difference in the fluorescence intensity is due to the complexation of Fe2+ ion with receptor1. The quenching in intensity proves that the receptor1 has the ability to attract Fe2+ ion from spiked water sample. The recovery percentage of Fe2+ ion for B1, B2 is 96%, 99.3%, for T1, T2 is 100.7%, 98.8% and for D1, D2 is 101.3%, 99.5% respectively (Table 1). The results proved that receptor1 could be practically applicable for selective detection of Fe2+ ion in real water sample analysis.
Table 1. Fe2+ ion recovered from real water sample spiked with Fe2+ ion using receptor1.
CIT Water
Sample
|
Fe2+ added (µM)
|
Fe2+ analysed from fluorescence spectra
(µM)
mean[a] ± SD[b]
|
Recovery (%)
|
Relative error (%)
|
|
|
|
|
|
Borewell water 1
Borewell water 2
|
3.0
|
2.88 ± 0.15
|
96.0
|
-4.0
|
6.0
|
5.96 ± 0.05
|
99.3
|
-0.7
|
Tap water 1
Tap water 2
|
3.0
|
3.02 ± 0.05
|
100.7
|
0.7
|
6.0
|
5.93 ± 0.03
|
98.8
|
-1.2
|
RO drinking water 1
RO drinking water 2
|
3.0
|
3.04 ± 0.14
|
101.3
|
1.3
|
6.0
|
5.97 ± 0.06
|
99.5
|
-0.5
|
|
a mean of three measurement.
b standard deviation.
Molecular Logic Gate
The application of molecular logic gates has attracted the scientist in sensor field [46, 47]. The reversible property of receptor1 helped to construct a molecular logic gate using two binary inputs. The absence and presence of Fe2+ and EDTA was represented by ‘0’ and ‘1’ (i.e. presence of Fe2+ and EDTA as 1 and the absence of Fe2+ and EDTA as 0). The output is monitored at 420 nm. Based on the change in fluorescence intensity the truth table was constructed. The output ‘0’ denotes the quenching intensity (fluorescence-off) and ‘1’ denotes the enhanced intensity (fluorescence-on). On the basis of truth table the INHIBIT logic gate was constructed as shown in Figure 9. Thus the addition of Fe2+ to receptor1 shows the weak fluorescence and on adding EDTA to receptor1-Fe2+ complex the fluorescence intensity is enhanced (Table 2).
Table 2. Truth table of INHIBIT logic gate
Input 1
(Fe2+)
|
Input 2
(EDTA)
|
Output
(λem 420nm)
|
0
|
0
|
1
|
1
|
0
|
0
|
0
|
1
|
1
|
1
|
1
|
1
|
|
Molecular docking Studies
Protein-ligand interaction plays a significant role in structure based drug designing. Molecular docking technique reveals the interaction between the ligand and protein [48]. The synthesized receptor1 acts as a ligand and it is subjected to molecular docking using Schrodinger maestro software to assess its interactions towards protein. The performance of receptor1 was high when it is docked with NUDT5, a macromolecule that act as a silence hormone signaling in breast cancer (PDB IB: 5NWH) [49]. The hydroxyl group of receptor1 interacts with the side chain residue ASP274. The hydrophobic interaction was noted between the thaizole moiety and benzene ring of the schiff base interacting with the amino acid residues such as LEU178, VAL174, LEU169, LEU164, PHE144, VAL147, LEU210, LEU208 and ALA273(Fig. 11). The docking score of receptor1 with protein is -5.23 kcal mol-1. The docking score of net negative energy represents a stable complex. The stable complex between receptor1 and amino acid residues are held by means of electrostatic interaction. Such interactions are due to the distribution of asymmetric charges in the receptor1 molecule containing heteroatoms such as N, O, and S. The docking result indicates that the receptor1 has shown good affinity towards NUDT5 target. Thus, receptor1 can act as an inhibitor for blocking NUDT5 activity.
Molecular keypad lock
The excellent reversible property helped us to propose a sequence of code for molecular keypad to lock. Three different chemical input was given as receptor1 ‘R’, Fe2+ metal ion ‘F’ and EDTA ‘E’. The six feasible input fusions are RFE, REF, FRE, FER, ERF, and EFR. In these combination, RFE resulted for enhanced fluorogenic output whereas the other combination such as REF,FRE,FER,ERF,EFR result in quenching intensity as output as shown in Figure 10. The keypad contains the alphabet from A to Z and the password RFE unlocks the keypad. At the same time, the other codes results for wrong password. Due to this reason, this type of sensing will safeguard the molecular level information.