Atul Katarkar

ESIPT blocked CHEF based differential dual sensor for Zn2+ and Al3+in a pseudo-aqueous medium with intracellular bio-imaging applications and computational studies

A novel 3-hydroxymethyl-5-methylsalicylaldehydenaphthyl-hydrazone (H3SAL-NH) exhibits ESIPT behaviour due to proton transfer from the phenolic OH group to the azomethine N atom in the excited state. Through this ESIPT behaviour together with cis–trans isomerization of the azomethine group, the free ligand becomes very weakly fluorescent. However, in the presence of Zn2+ and Al3+ the ESIPT and isomerization are blocked due to coordination to the metal ions thereby causing turn on fluorescence for Al3+ and Zn2+. Moreover, Zn2+ can easily be displaced from the [H2SAL-NH–Zn2+] complex by Al3+ thereby enhancing the differential selectivity for Al3+ over Zn2+. This probe was found to be selective for Al3+ over Zn2+ in the presence of Na2H2EDTA, under both intra- and extracellular conditions. The LODs for Zn2+ and Al3+ were determined by 3σ methods and were found to be 3.1 nM and 0.92 nM, respectively. Thus, the differentially selective turn-on fluorescence behaviour of H3SAL-NH for Zn2+ and Al3+ is based on the combined blocking of ESIPT and CN isomerization, and a chelation-enhanced fluorescence (CHEF) effect. The coordination modes of the complexes were investigated through spectroscopic and computational studies. H3SAL-NH also exhibits good photostability and very low cytotoxicity and is useful for fluorescence imaging of Zn2+ and Al3+ ions in live HepG2 cells.

A novel rhodamine-3,4-dihydro-2H-1,3-benzoxazine conjugate as a highly sensitive and selective chemosensor for Fe3+ ions with cytoplasmic cell imaging possibilities

A novel, highly sensitive and selective fluorescent chemosensor ‘rhodamine-3,4-dihydro-2H-1,3-benzoxazine’ [RH-BZN (1)] has been synthesized and characterized by single crystal X-ray diffraction and other physicochemical techniques. In 3:7 water:MeCN (v/v) at pH 7.2 (10 mM HEPES buffer, μ = 0.05 M LiCl) it selectively recognizes Fe3+ through 1:1 complex formation resulting in a 240-fold fluorescence enhancement and a binding constant (Kf) of 1.50 × 104 M−1. The otherwise non-fluorescent spirolactam form of the probe results in dual changes in absorbance and fluorescence arising out of opening of the spirolactam ring through coordination to Fe3+ ions. This probe could suitably be employed for cytoplasmic intracellular imaging of Fe3+ without notable cytotoxicity. The reversible binding of RH-BZN to Fe3+ was confirmed by reacting with tetra-n-butylammonium fluoride both in extra- and intra-cellular conditions.

A rhodamine based fluorescent trivalent sensor (Fe3+, Al3+, Cr3+) with potential applications for live cell imaging and combinational logic circuits and memory devices

A sensor (HL5) based on rhodamine 6G–en and 3-(3,5-dimethyl-pyrazol-1-ylmethyl)-2-hydroxy-5-methyl-benzaldehyde (HL4) has been developed for a highly sensitive and selective CHEF based recognition of trivalent metal ions M3+ (M = Al, Fe and Cr) over mono- and di-valent and other biologically abundant trivalent metal ions with prominent enhancement in absorption and emission intensities. A large enhancement of fluorescence intensities for Fe3+ (21 fold), Al3+ (14 fold) and Cr3+ (10 fold) was observed upon addition of 1.8 equivalents of these metals into the probe in methanol/H2O (1 : 1, v/v, pH 7.2) with the possibility of naked eye detection. The corresponding Kf values were evaluated to be 6.7 × 104 M−1 (Fe3+); 8.2 × 104 M−1 (Al3+) and 6.0 × 104 M−1 (Cr3+). The quantum yields of HL5 and [HL5–Fe3+] and [HL5–Cr3+] and [HL5–Al3+] complexes in methanol/H2O (1 : 1, v/v, pH 7.2) are found to be 0.013, 0.290, 0.120, and 0.158, respectively, using rhodamine-6G as the standard. The LOD for Fe3+, Al3+ and Cr3+ were determined by 3σ methods with values 0.29, 0.34 and 0.31 μM, respectively. An arsenate ion snatches Al from the Al–HL5 complex and quenches its fluorescence via its ring closed spirolactam form. Advanced level molecular logic devices using the inputs 2 and 4 and memory devices, have been constructed. The low cytotoxicity and large enhancement in fluorescence intensity of HL5 upon complexation with M3+ metal ions make the probe suitable for bio-imaging of M3+ (M = Al, Fe and Cr) in living cells and native cellular iron pools.

A novel 8-hydroxyquinoline-pyrazole based highly sensitive and selective Al(III) sensor in a purely aqueous medium with intracellular application: experimental and computational studies

A new 8-hydroxyquinoline-pyrazole based highly sensitive and selective Al3+ sensor, 8Q-NH-Pyz (H2L3), was found to exhibit a turn-on fluorescence enhancement (FE) as high as 157 fold with Kd = (1.76 ± 0.06) × 10−5 M. The 1:1 binding stoichiometry was revealed from the linear fit of (Fmax − F0)/(F − F0) vs. 1/[Al3+] of the fluorescence titration data which was further substantiated by Jobs method and HRMS studies. The LOD determined by 3σ methods was found to be 4.29 nM and quantum yields were determined to be 0.002 and 0.28 for the ligand and its Al3+ complex, respectively. The tentative coordination environment in the [Al(L3)(H2O)]+ complex was delineated by DFT calculations. TDDFT calculations reveal spectral features comparable to the experimental ones. This constitutes the first report on the fluorescence sensing of Al3+ and hence F− in a purely aqueous medium.

A novel pyrene-2-(pyridin-2-ylmethylsulfanyl)ethylamine based turn-on dual sensor for Al3+: experimental and computational studies

A new pyrene based highly sensitive and selective Al3+ sensor, pyrene-2-(pyridin-2-ylmethylsulfanyl)-ethylamine (PP), was found to exhibit a turn-on fluorescence enhancement (FE) as high as 7.4 fold with Kd (2.55 ± 0.10) × 10−4 M and n = 1. This probe binds reversibly with Al3+ in the presence of H2EDTA2−, both under intra- and exctracellular conditions. LOD determined by 3σ methods was found to be 15 nM while LOQ = 52.8 nM. The tentative coordination environment in the Al3+–PP complex was delineated by DFT calculations both on the free ligand and complex. The TDDFT calculations reveal spectral features comparable to the experimental ones.

Comparative evaluation of genotoxicity by micronucleus assay in the buccal mucosa over comet assay in peripheral blood in oral precancer and cancer patients

Early detection and quantification of DNA damage in oral premalignancy or malignancy may help in management of the disease and improve survival rates. The comet assay has been successfully utilised to detect DNA damage in oral premalignant or malignancy. However, due to the invasive nature of collecting blood, it may be painful for many unwilling patients. This study compares the micronucleus (MN) assay in oral buccal mucosa cells with the comet assay in peripheral blood cells in a subset of oral habit-induced precancer and cancer patients. For this, MN assay of exfoliated epithelial cells was compared with comet assay of peripheral blood leucocytes among 260 participants, including those with oral lichen planus (OLP; n = 52), leukoplakia (LPK; n = 51), oral submucous fibrosis (OSF; n = 51), oral squamous cell carcinoma (OSCC; n = 54) and normal volunteers (n = 52). Among the precancer groups, LPK patients showed significantly higher levels of DNA damage as reflected by both comet tail length (P < 0.0001) and micronuclei (MNi) frequency (P = 0.0009). The DNA damage pattern in precancer and cancer patients was OLP < OSF < LPK < OSCC, and with respective oral habits, it was multiple habits > cigarette + khaini > cigarette smokers > areca + khaini > areca. There was no significant difference in the comet length and MNi frequency between males and females who had oral chewing habits. An overall significant correlation was observed between MNi frequency and comet tail length with r = 0.844 and P < 0.0001. Thus, the extent of DNA damage evaluation by the comet assay in peripheral blood cells is perfectly reflected by the MN assay on oral exfoliated epithelial cells, and MNi frequency can be used with the same effectiveness and greater efficiency in early detection of oral premalignant conditions.

A rhodamine embedded bio-compatible smart molecule mimicking a combinatorial logic circuit and 'key-pad lock' memory device for defending against information risk

Organic molecules with the possibility of logic operations are highly useful building blocks for the development of molecule-based “intelligent” devices for information processing applications. We have designed herein a very simple bio-friendly chemosensor (LC) equipped with a rhodamine fluorophore moiety. This probe showed a chromo-fluorescence response profile for Al3+ but a colorimetric response for Cu2+ metal. The absorption responses of LC caused by these metal ions along with the “OFF–ON–OFF” fluorescence behavior of an LC–Al3+ complex towards EDTA were employed for the development of a three-input and one output combinatorial molecular system. Interactions of the mentioned metal ions with LC in controlled sequential experiments gave fluorescence responses, enabling us to fabricate a ‘key-pad-logic’ function. So, a single molecular system performing such multiple ‘Boolean’ operations not only simplifies the complexity of a chemical driven ‘Intelligence’ device but also enriches the security of such a device against information invasion due to the sequence controlled sensor–analyte interactions and may find potential applications in biocompatible molecular logic platforms.

A Smart Molecule for Selective Sensing of Nitric Oxide: Conversion of NO to HSNO; Relevance of Biological HSNO Formation

A smart molecule, QT490, containing thiosemicarbazide moiety acts as a highly selective turn-on in vitro NO sensor through the unprecedented NO-induced transformation of thiosemicarbazide moiety to 1,3,4-oxadiazole heterocycle with the concomitant release of HSNO, thereby eliminating any interference from various endogenous biomolecules including dehydroascorbic acid, ascorbic acid, etc. The kinetic studies of the reactions between QT490 and NO provide a mechanistic insight into formation of HSNO/RSNO from the reaction between H2S/RSH and NO in the biological system. This novel probe is non-cytotoxic, cell permeable, water-soluble, and appropriate for intracellular cytoplasmic NO sensing with the possibilities of in vivo applications.

A thiosemicarbazone based chemo and fluorogenic sensor for Zn2+ with CHEF and ESIPT behaviour: computational studies and cell imaging application

We report herein the development of a novel, diformyl-p-cresol (DFC)–thiosemicarbazide (TS) based sensor (DFC–TS) that selectively and sensitively recognizes Zn2+ by both UV-Vis and fluorescence methods. The gradual addition of Zn2+ to a solution of the ligand developed a new absorption band at 430 nm, while the bands at 370 and 316 nm gradually decrease generating one well defined isosbestic point at 390 nm exhibiting ∼17 fold turn-on fluorescent enhancement (FE). When we plot absorbance (at 430 nm) vs. [Zn2+] there is a gradual increase in absorption with [Zn2+], becoming saturated at ∼1 equivalent of Zn2+ and then again it increases with the increase in [Zn2+] and ultimately becomes saturated at ∼2 equivalents of added Zn2+. This clearly demonstrates that the Zn2+ binding event to the ligand occurs in two steps, one at a time. Non-linear least-squares computer-fitting of these data gives the parameters: K′f1 = (9.70 ± 5.51) × 105 M−1, n = (1.28 ± 0.05) for the first step and K′f2 = (1.11 ± 0.65) × 105 M−1 and n = (1.01 ± 0.06) for the second step. So far, this study provides the opportunity where we have successfully, for the first time, determined the stepwise formation constants; though they have values of the same order of magnitude. The ground state geometries of DFC–TS, both enol and keto forms and [Zn(DFC–TS)(OAc)], [Zn(DFC–TS)(OAc)]−, and [Zn2(DFC–TS)(OAc)2] were optimized using the Gaussian-03 suit program and bond distances of all species are in reasonable agreement with the reported values.

Dual channel selective fluorescent detection of Al3+ and PPi in mixed aqueous media: DFT studies and cell imaging applications

A new, easily synthesizable chemosensor, DFC-EN-p-Ph-NO2, derived by the Schiff base condensation between 2,6-diformyl-p-cresol and N-(4-nitrophenyl)ethylenediamine, with potential N4O donor atoms was found to act as a dual channel (colori- and fluorimetric) sensor towards Al3+ and PPi emitting at 486 nm (blue region) and 534 nm (green region), respectively in MeOH–H2O (8 : 2, v/v) at pH 7.2 (10 mM HEPES buffer), μ = 0.05 M (LiCl), temperature 25 °C. The binding stoichiometries and formation constants of the sensor towards both Al3+ and PPi were determined by the combined UV-Vis and fluorescence titrations and Jobs method, and confirmed by MS (m/z) studies. The corresponding detection limits as calculated by the 3σ method are: 7.55 μM and 3.34 μM. The most interesting part of this study is that on addition of 230 μM PPi to an ensemble of DFC-EN-p-Ph-NO2−Al3+ (20 μM Ligand and 380 μM Al3+) the fluorescence is totally quenched but on further addition of PPi a new emission peak appears at 534 nm. All biologically relevant metal ions and toxic heavy metals did not interfere with the detection of Al3+ ion. Its bio-compatibility with respect to its good solubility in mixed organo-aqueous media (MeOH–H2O) and cell permeability with no or negligible cytotoxicity provide good opportunities towards in vitro cell imaging studies of these ions. In particular, the fluorescent detection of PPi was not interfered by the presence of 400 μM of ATP or Pi although most reported PPi sensors that work in aqueous solution displayed cross-sensitivities toward ATP or Pi. The obvious excellent sensing capability of DFC-EN-p-Ph-NO2 towards PPi and Al3+ was further scrutinized in HCT116 cell lines without much cytotoxicity. The modes of 1 : 1 binding of DFC-EN-p-Ph-NO2 towards Al3+ and PPi were delineated by DFT calculations.