Due to the many advantages and potential of the chemosensors, they have been used in various fields. As soon as the serum encounters the sensor molecules, Na+ in the serum binds to the appropriate fraction of the sensor molecules.
2,6-Diformyl-4-methylphenol was prepared by adapting the literature method.2.1 Absorption spectra were recorded on a Perkin-Elmer Lamda-25 UV−vis spectrophotometer using quartz cuvettes with a path length of 10 mm in the range 250−700 nm wavelength. The reversible behavior of L1 after the successive addition of Zn2+ and PPi was performed in the same mixed solvent.
Detection Limit for Zn 2+ ion and PPi 14
Detection of PPi in PCR Experiment 15
The template DNA was initially subjected to denaturation for 2 min at 94°C, followed by amplification cycles in a programmable thermal cycler (CG Palmcycler). To visualize the PCR products, the generated amplicons corresponding to different samples (5 to 35 cycles) were also subjected to agarose gel (0.8%) electrophoresis.2.4.
Detection of PPi in PCR Experiment and Estimation of Bacterial Cell Numbers 15
Separate set of samples in triplicate were subjected to varying durations of PCR ranging from 5 to 35 cycles. To determine the level of PPi generated in the PCR experiments, a 10 µL aliquot of PCR mixture from each sample (5 to 35 cycles) was then separately added to 400 µL 10 mM HEPES buffer (pH 7.4 ) added containing 5 µM L1. 2Zn complex and the fluorescence emission intensity was measured at 540 nm.
Detection Limit for Zn 2+ ion 17
To obtain the slope, the emission intensity ratio at 550 nm was plotted against the concentration of Zn2+. The limit of detection was calculated using Equation 2.2, where σ is the standard deviation of the blank measurement and k is the slope between the ratios of emission intensity versus [Zn2+].
Cytotoxicity assay for L 2 and L 2 -Zn complex 17
UV−Vis and fluorescence spectral studies with L 3 18
All the spectroscopic experiments of the cations were performed in aqueous HEPES buffer medium (1 mM, pH 7.4) containing 0.33% DMSO. In selectivity experiments, the test samples were prepared by interacting appropriate amounts of the cation stock in 2 mL of L3 solution (2×10−5 mol L−1).
Evaluation of the binding constant 18
Spectroscopic studies of L3 in the presence of various anions were performed in acetonitrile medium containing 0.33% DMSO. For all samples, the spectra were recorded after 1 min after the addition of the ions.
Detection Limit of L 3 for different ions 19
X-ray crystallography 19
Cytotoxicity assay 20
Then, the MTT solution was carefully removed and the insoluble formazan-colored product was dissolved in DMSO and its absorbance was measured in a microtiter reader (Infinite M200, TECAN, Switzerland) at 550 nm. Data analysis and calculation of standard deviation was performed with Microsoft Excel 2010 (Microsoft Corporation, USA).
Cell imaging studies 20
HeLa cells treated with DMSO or the metal salts were also included in parallel sets as a control. The selective binding of L4 to Al3+ among all other metal ions was also studied by fluorescence emission spectroscopy of the solution of L molL−1) in the absence and presence of an excess (10 eq.) of each of the metal ions in mixed solvent . .
Detection Limit for Al 3+ ion 21
Before the MTT assay, cells were seeded in 96-well tissue culture plates (approximately 104 cells per well) and incubated with different concentrations of compound L4 and L4–Al3+. Then the MTT solution was removed and the insoluble colored formazan product was solubilized in DMSO and its absorbance was measured in a microtiter plate reader (Infinite M200, TECAN, Switzerland) at 550 nm.
Interaction with calf thymus DNA (CT-DNA) and tracking DNase I activity 23
After the specified incubation time, the reaction mixtures were added in separate sets to 1.0 mL solution of L4-Al3+ complex (2.5 M) made in 5.0 mM HEPES buffer: MeOH (3:2) and the contents were thoroughly mixed by inverting the tubes . Next, the fluorescence emission intensity of the samples was measured at an excitation wavelength of 640 nm and the emission spectra collected in scanning mode from 660 nm to 800 nm.
Synthesis and characterization of the compounds 23
Synthesis of L2 23
Synthesis of picolinichydrazide 23
Synthesis of N-ethyl-2, 3, 3-trimethyl-3H-indolium iodide (1) 25
Synthesis of 2 25
Synthesis of Cy.7.Cl (3) 26
Changes in the fluorescence emission spectra of L1.2Zn (10 μM) in the mixed solvent after adding (a) different anions and (b) increasing concentration of PPi. Changes in the emission spectra of L3 (25 μM) (a) with the addition of different anions (b) with the stepwise addition of F.
Therefore, the response of L1 to Zn2 + to other metal ions was investigated in the presence of each of those metal ions (5 eq.) mimicking the physiological condition. It was indeed encouraging to note that the fluorescence of L1 induced by Zn2+ was preserved in each of the cases clearly suggesting the high selectivity of L1 towards Zn2+ even in the presence of these metal ions (Fig. A3.2a.).
Changes in the UV-visible absorption spectra of L1.2Zn (10 M) after addition of (a) various anion solutions (10 eq), (b) increasing amount of PPi. The changes in the color of the solutions of L1.2Zn complex in the presence of different anions were studied under UV light (365 nm).
Therefore, it can be assumed that the selective sensing of PPi is probably due to the stronger binding affinity of the PPi anion with the dinuclear complex and the regeneration of free L1 takes place via the formation of the L1.Zn.P2O7 complex.
Molecular Logic Gate Based on the Reversible Behavior of L 1 with Sequential
Aliquots of the PCR reaction mixture from different numbers of cycles were also separately reacted with the L1.2Zn complex and the level of PPi produced in the samples was ascertained by measuring the fluorescence intensity of the complex. Inset: % fluorescence quenching of L1.2Zn complex after interaction with PCR reaction products corresponding to samples B–H.
Estimation of Bacterial Cell Numbers 52
Fluorescence intensity of the L1.2Zn complex after interaction with PCR amplicons obtained after 10 cycles from different cell numbers of E.coli MTCC 433. Inset: % fluorescence quenching of the L1.2Zn complex after interaction with PCR reaction products obtained after 10 cycles from different cell numbers .
Conclusions 54
The gray bars represent the emission intensities of L3 in the presence of cations of interest (5 equiv). Titration of L4 with gradual addition of Al3+ resulted in a linear enhancement of the emission.
Absorption spectroscopic studies 62
In the case of L2, the conjugation breaks after the –C=O group, so the absorption peak is at a much shorter wavelength (367 nm) compared to the absorption peak at 450 nm. The absorption titration experiment also showed that the change in the absorption spectra became minimal after the addition of two equivalents of metal ions (Fig. 4.1b inset), suggesting a 1:2 bond stoichiometry between L2 and Zn2+.
After the addition of two equivalents of Zn2+ ion, the change in the emission spectra became. A change in the fluorescence emission from colorless to yellow was observed under UV lamp after the addition of Zn2+ to the receptor solution (Fig. 4.2a inset).
The gray bars represent the change in emission upon subsequent addition of Zn2+ to the above solution. Collectively the 1H-NMR titration strongly suggested the formation of a hydroxo-bonded complex between L2 and Zn2+ resulting in deprotonation of the hydroxyl group and the pyridyl nitrogen atom coordinating to Zn2+ causing displacement of the pyridyl hydrogen atoms.
ESI-MS Experiment 66
The reduction in the intensity of the hydroxyl group (-OH) indicated deprotonation as a result of the interaction with Zn2+ and after the addition of two equivalents of Zn2+ ion, the peak of the hydroxyl group was erased. The Schiff base hydrogen atom and the hydrogen atoms in the DFMP ring also underwent field shifts, while very little change was observed for the amide proton (-NH) compared to the other protons within the system.
Mechanism of Zn 2+ Sensing 66
Theoretical Calculations of L 2 and L 2 -Zn Complex 67
These HOMO and LUMO energy diagrams revealed that the energy gap of the dinuclear zinc complex of L2 (two zinc ions are connected through the Oxo bridge) was reduced compared to the ligand alone (Fig. This perhaps led to the appearance of red-shifted (25 nm) fluorescence emission band after chelating the ligand with two Zn2+ ions in the proposed manner.
Selected orbitals and their corresponding energies for both L2 and its Zn complex were provided in supporting information (Table.S1 and S2 in supporting information) that may have played an important role in the optical spectral outcome.
Conclusion 69
The gray bars represent the emission intensity of L4 in the presence of the cations of interest (5 eqv). The interaction with CT-DNA resulted in a nominal reduction of the fluorescence intensity of the L4-Al3+ complex (Fig. 6.5).
The titration experiment with Zn2+ gave analogous results as in the case of Al3+, although a difference of higher magnitude (Fig. 5.2c). Interestingly, a visual color change from colorless to light yellow and deep yellow was observed with Al3+ and Zn2+, respectively (Fig. 5.2b inset).
Job's plot obtained from the titration experiments yielded 1:2 stoichiometry for both Al3+ and Zn2+ (Fig. A5.1a, A5.2a). The selectivity of L3 towards Al3+ and Zn2+ was also established by experiments in the presence of competing metal ions (Fig. A5.3a, A5.3b).
Titration experiments were also performed with gradual addition of Al3+ and Zn2+ to the L3 solution. Changes in absorption spectra of L3 (25 μM) with (a) addition of different anions (b) increasing addition of F.
This phenomenon was validated by adding a small volume of water/methanol, yielding a colorless solution.
Initially, the phenolic –OH group was deprotonated and a downfield shift along with reduction in the intensity of the –NH proton was observed (=0.055 ppm). So, due to deprotonation of both the phenolic OH and -NH groups, sharp changes were observed in the case of fluoride in both absorption and emission spectroscopy.
Plausible Mechanism of sensing 83
The imine proton also faced a downward shift of =0.116 ppm, while the protons of the quinoline ring were shifted upward, but the shifts () were small compared to other changes.
HeLa cells incubated with only L3 did not display any fluorescence, while bright green fluorescence manifested in the cells upon addition of the target metals (Fig. 5.7.). It was also evident that the HeLa cells retained their morphological characteristic during the cell imaging studies (Fig. 5.7), reiterating the non-toxic nature of the developed receptor.
Conclusion 84
It was also noted that the fluorescence emission spectra of the receptor L4 remained unaffected in the presence of Fe3+ (Fig. 6.2a), although a marginal change in the absorption spectra of L4 could be observed upon interaction with Fe3+ (Fig. 6.1a). . The orange bars represent the changes in the emission intensities of L4-Al3+ complex upon the subsequent addition of different phosphates to the above solution.
Background and focus of the chapter 91
To overcome this barrier, Al3+-selective fluorescent probes with absorption and emission in the near-infrared (NIR) region (650–900 nm) are expected to be highly desirable due to reduced potential for photodamage to biological samples, increased number of sample penetration and minimal background auto-fluorescence, which would greatly enhance bioimaging studies. The receptor can selectively detect Al3+ among other trivalent and biologically relevant cations via a remarkable fluorescent emission signal captured in the.
It may also be mentioned here that a visual change in the color of a solution of L4 from colorless to purple was observed during the titration experiment (Fig. 6.1b, inset), which made the naked eye detection of Al3+ in solution. Changes in the absorption spectra of L4 (in mixed solvent medium methanol: HEPES buffer (2:3) medium (5 mM, pH 7.4) containing 0.33% DMSO) (a) with the addition of different metal ions and (b) with gradual addition of Al3+.
Changes in the emission spectra of L4 (in mixed solvent medium methanol: HEPES buffer (2:3) medium (5 mM, pH 7.4) containing 0.33% DMSO) after addition of (a) different metal ions and (B) after gradual addition of Al3+. The black lines represent the change in emission that occurs after the subsequent addition of Al3+ to the above solution.
ESI-MS Experiment 95
Plausible mechanism of Al 3+ sensing 95
On the other hand, the visual color change can be attributed to the activation of the ICT process. Furthermore, after merging the DAPI-stained and L4-Al3+-stained images, it was evident that the red fluorescence emission originated from L4-Al3+.
It is significant to mention that the characteristic morphology of HeLa cells was preserved during imaging studies, which reiterated the non-toxic nature of the L4 probe.
Conclusion 98
The black triangles are the fluorescence intensities of L4 at different pH while the black square blocks are the fluorescence intensities of L4 with the addition of Al3+. a) MTT assay to determine the cytotoxic effect of L4 complex and L4-Al complex on HeLa cells. Interestingly, upon interaction with DNA in solution, the L4-Al3+ ensemble tracked DNase activity in solution through a systematic reduction in fluorescence emission intensity.
