Atul Kumar Dwivedi

Aqueous polyfluorene probe for the detection and estimation of Fe3+ and inorganic phosphate in blood serum

A novel anionic polyfluorene derivative, poly(9,9-bis(6′-sulfate)hexyl) fluorene-alt-1,4-phenylene sodium salt (P1) is synthesized. P1 exhibits exemplary activity towards the selective detection of Fe3+ and phosphates (Pi) under physiological conditions. On binding to Fe3+, exceptional fluorescence quenching of P1 occurred, demonstrated by a >97% reduction in the fluorescence intensity. Furthermore, the P1–Fe3+ assay is highly selective for inorganic phosphate (Pi) anions at biological pH values, observed by complete fluorescence dequenching and confirmed through a >95% fluorescence enhancement. In order to validate its diagnostic potential, this assay was employed to monitor the Pi levels in a competing biological environment like blood serum. At pH 7.4 this assay showed a high specific activity to detect Pi in the bioassay environment, observed by the unique enhancements in fluorescence intensities for varying and low Pi concentrations. Since this assay performed Pi detection at very low concentrations we utilized it successfully for the fluorometric estimation of Pi in blood serum with high accuracy and within short duration. This remarkable ability of P1 to accomplish in situ monitoring and estimation of indispensable biological targets like Fe3+ and Pi rapidly and in label-free conditions, corroborates the extension of this assay system for clinical application.

Assembly Modulation of PDI Derivative as a Supramolecular Fluorescence Switching Probe for Detection of Cationic Surfactant and Metal Ions in Aqueous Media

We report an amphiphilic perylene diimide (1), a bimolecular analog of l-3,4-dihydroxyphenylalanine (L-DOPA), as a reversible fluorescence switching probe for the detection and sensing of cationic surfactants and Fe(3+)/Cu(2+) in an aqueous media respectively by means of host-guest interactions driven assembly and disassembly of 1. Photophysical studies of 1, going from dimethyl sulfoxide (DMSO) (State-I) to pure aqueous medium (State-II), suggested the formation of self-assembled aggregates by displaying very weak fluorescence emission along with red shifted broad absorption bands. Interestingly, the cationic surfactant cetyltrimethylammonium bromide (CTAB) could disassemble 1 in miceller conditions by restoring bright yellow fluorescence and vibronically well-defined (Franck-Condon progressions A0-0/A0-1 ≈ 1.6) absorption bands of 1 over other neutral and anionic surfactants (State-III). Owing to the metal chelating nature of L-DOPA, 1 was able to sense Fe(3+) and Cu(2+) among a pool of other metal ions by means of fluorescence switching off state, attributed to metal interaction driven assembly of 1 (State-IV). Such metallosupramolecular assemblies were found to reverse back to the fluorescence switching on state using a metal ion chelator, diethylenetriaminepentaacetic acid (DTPA, State-III), further signifying the role of metal ions toward assembly of 1. Formation of assembly and disassembly could be visualized by the diminished and increased yellow emission under green laser light. Further, the assembly-disassembly modulation of 1 has been extensively characterized using infrared (IR), mass spectrometry, microscopy and dynamic light scattering (DLS) techniques. Therefore, modulation of the molecular self-assembly of PDI derivative 1 in aqueous media (assembled state, State-II) by means of host-guest interactions provided by micellar structures of CTAB (disassembled state, State-III), metal ion (Fe(3+) and Cu(2+)) interactions (assembled state, State-IV) and metal ion sequestration using DTPA (disassembled state, State-III) is viewed as a supramolecular reversible fluorescence switching off-on probe for cationic surfactant CTAB and Fe(3+)/Cu(2+).

Development of solution, film and membrane based fluorescent sensor for the detection of fluoride anions from water

The synthesis and application of a neutral polymer, poly(1,4-bis-(8-imidazole-octyloxy)-benzene) (PPI), is performed by economical and simple reaction steps. The PPI polymer demonstrates exemplary activity to be used as a film on a TLC plate, or as a membrane by blending it with a desired polymer or in a solution phase to detect fluoride anions from contaminated water in the presence of competing anions at ppb levels easily and rapidly. This polymer PPI works on the simple displacement principle where fluorescence turn-on/turn-off are observed as signals. On selectively binding Cu2+ it displays extraordinary fluorescence quenching, resulting in >97% reduction in the fluorescence intensity. This effect could be visualized in solution phase, on a TLC plate and on a blended polymer membrane. Furthermore, the fluorescence of this PPI–Cu2+ assay showed 81% enhancement on selectively binding F− anions in contaminated water in the presence of other competing anions with higher positive free energies of hydration. Polymeric systems with such robust fluorescence dequenching activity are novel, providing a unique platform for detection and possible removal of fluoride anions. To validate this potential, two experiments were performed: (a) preparation of a film on a TLC plate and (b) preparation of a membrane by mixing 1% PPI in polystyrene and casting as a membrane film of desired shape and thickness. Our results confirm that PPI–Cu2+ films and membranes described above have the highest specific activity to sense fluoride in a competitive environment, observed by the unique enhancements in fluorescence intensities at varying and extremely low quantities of 1 ppm, 10 ppm and 50 ppm of fluoride. The detection limit of fluoride in contaminated water for the TLC plate and membrane methods was very low and was in the range of 2.5–10.0 ppb. We have further used these methods for the detection of fluoride in natural ground water samples and ascertained the percentage of fluoride.

Interaction of heme proteins with anionic polyfluorene: insights into physiological effects, folding events, and inhibition activity.

Because of the toxicity caused by the heme redox-active iron proteins, their elevated levels, localization, and accumulation in the brain, many forms of neurodegenerative diseases, such as Alzheimers disease, Parkinsons disease, and Huntingtons disease, occur as a result of which the brain becomes vulnerable to oxidative stress, ultimately resulting in neuronal death. An anionic water-soluble conjugated polyfluorene derivative poly(9,9-bis(6-sulfate hexyl) fluorene-alt-1,4-phenylene) sodium salt (P1) that binds Fe³⁺ proteins with very high selectivity and sensitivity is reported here. The photophysical properties of P1 were modified by the interaction with ferric heme-containing proteins cytochrome c (Cc), methemoglobin (MetHb), and hemin. P1 was found to be highly sensitive toward Fe³⁺ heme proteins as compared to nonmetalloproteins. We observed that the respective activities of ferric heme proteins were inhibited and proteins were unfolded, due to modification in their heme microenvironment in the presence of the polymer P1. The observations reported in this article provide the first example for the use of a water-soluble conjugated polymer in applications, such as (1) to detect small quantities of iron proteins in aqueous medium/physiological condition with the highest K(sv) values of 2.27 × 10⁸ M⁻¹ for Cc, 3.81 × 10⁷ M⁻¹ for MetHb, and 5.31 × 10⁷ M⁻¹ for hemin; (2) to study the physiological effects of heme metalloproteins; (3) to visualize the folding events in real time; and (4) the inhibition activity of metalloproteins can be selectively studied using a conjugated polymer based assay system rapidly without interference from nonmetalloproteins at biological pH. All this is achieved by generating optical events, taking advantage of the bright fluorescence of anionic polyfluorene P1 in this case, that can be observed and monitored by modification in the absorption and emission color in real time.

A fluorescence turn on trypsin assay based on aqueous polyfluorene

A new method based on the electrostatic interaction of a novel anionic water soluble polymer P1 with a positively charged polypeptide Arg6 was developed for a continuous and real time turn on assay for the enzymatic activity of trypsin under alkaline conditions with a limit of detection of 0.17 nM. This method was also able to screen the inhibitors of trypsin. P1 fluorescence intensity was significantly decreased by the positively charged Arg6 due to the electrostatic interaction, whereas the enzymatic action recovered P1 fluorescence due to the fragmentation of Arg6 into small positively charged fragments and these were unable to quench the P1 fluorescence. Therefore, by triggering the fluorescence intensity change, it was possible to assay the enzymatic activity. Use of water soluble conjugated polymer P1 and no labeling on the substrate enhances the utility of this method significantly.

Sensitive detection of acid phosphatase enzyme and screening of inhibitors using an anionic polyfluorene derivative

An assay method, that includes a ferric iron bound to a novel anionic water soluble polymer (P1), is developed, for the continuous and real time fluorescent amplification detection of acid phosphatase (ACP) enzyme activity under acidic conditions in nanomolar quantities. The ferric iron bound P1 offers a sensitive and rapid turn on assay for inorganic phosphate (Pi) selectively. The most frequently used phosphatase substrate p-nitro phenyl phosphate (p-NPP), which was inactive towards the dequenching of P1–Fe3+ fluorescence, has been utilized in the study as a model compound for the enzymatic hydrolysis and a very small concentration of enzyme in nanomolar regime was sufficient to generate a significant fluorometric change during enzymatic hydrolysis. Kinetic parameters were derived by observing the fluorometric changes of P1 during enzymatic hydrolysis providing vital information on the changes observed in the enzyme kinetics in a competitive environment. Additionally, this assay also offers high throughput screening of ACP inhibitors that may render the usefulness of this method in drug discovery.

Therapeutic Strategies to Prevent Alzheimer's Disease Pathogenesis Using A Fluorescent Conjugated Polyelectrolyte†

Toxic metals accumulation in brain has a significant role in the pathogenesis of Alzheimers disease (AD) by accelerating amyloid β (Aβ) peptide aggregation. Aβ has high affinity for iron and copper resulting in the generation of neurotoxic hydrogen peroxide, oxidative stress and free radical formation. Water-soluble conjugated polyfluorene derivative poly(9,9-bis(6-sulphate hexyl) fluorene-alt-1,4-phenylene) sodium salt (P1) binds Fe(3+) heme proteins selectively in cerebrospinal fluid (CSF), including ferritin in the Aβ fibrils and diminishes their accumulation. Hence, therapeutic strategies involving clearance of Aβ from brain plaques, metal removal, structurally modifying the aggregates, and preventing them from aggregating again into toxic polypeptides are vital strategies to control AD pathogenesis.

Exploration of Energy Modulations in Novel RhB-TPE-Based Bichromophoric Materials via Interactions of Cu2+ Ion under Various Semiaqueous and Micellar Conditions

Novel bichromophoric materials TR-A and TR-B consisting of an entirely new combination of TPE and RhB units were developed to explore the optimum conditions of energy modulations via pH variation and Cu(2+) interaction at various water contents of CH3CN. Interestingly, TR-A and TR-B, at 60 and 70% water contents, respectively, favored the optimum Cu(2+)-mediated energy modulations from TPE to RhB and thus achieve the brightest orange emissions of free RhB with complete disappearance of aggregation-induced emission (AIE) from TPE. Furthermore, various micellar conditions of triton-X-100, SDS, and CTAB were employed to adjust energy modulations of TR-A and TR-B at high water contents (at 80 and 90%, respectively). The incorporation of RhB into triton-X-100 micellar cavities disrupted AIE from TPE; thus, none of the energy modulations from TPE to RhB occurred even in the presence of Cu(2+) ion. Interestingly, the micellar conditions of anionic surfactant (SDS) favored the increased local concentration of Cu(2+) ions in the vicinity of scavangable RhB and facilitated the generation of noncyclic free RhB in situ via bright-orange emissions.