Pramod Kumar Verma

Bright, NIR-Emitting Au23 from Au25: Characterization and Applications Including Biolabeling

A novel interfacial route has been developed for the synthesis of a bright-red-emitting new subnanocluster, Au(23), by the core etching of a widely explored and more stable cluster, Au(25)SG(18) (in which SG is glutathione thiolate). A slight modification of this procedure results in the formation of two other known subnanoclusters, Au(22) and Au(33). Whereas Au(22) and Au(23) are water soluble and brightly fluorescent with quantum yields of 2.5 and 1.3 %, respectively, Au(33) is organic soluble and less fluorescent, with a quantum yield of 0.1 %. Au(23) exhibits quenching of fluorescence selectively in the presence of Cu(2+) ions and it can therefore be used as a metal-ion sensor. Aqueous- to organic-phase transfer of Au(23) has been carried out with fluorescence enhancement. Solvent dependency on the fluorescence of Au(23) before and after phase transfer has been studied extensively and the quantum yield of the cluster varies with the solvent used. The temperature response of Au(23) emission has been demonstrated. The inherent fluorescence of Au(23) was used for imaging human hepatoma cells by employing the avidin-biotin interaction.

Functionalized Au22 Clusters: Synthesis, Characterization, and Patterning

We synthesized fluorescent, porphyrin-anchored, Au(22) clusters in a single step, starting from well-characterized Au(25) clusters protected with glutathione (-SG) by a combined core reduction/ligand exchange protocol, at a liquid-liquid interface. The prepared cluster was characterized by UV/vis, photoluminescence, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy, elemental analysis, and matrix-assisted laser desorption ionization mass spectrometry. The absence of a 672 nm intraband transition of Au(25) and the simultaneous emergence of new characteristic peaks at 520 and 635 nm indicate the formation of the Au(22) core. An increase in the binding energy of 0.4 eV in Au 4f core-level peaks confirmed the presence of a reduced core size. Quantitative XPS confirmed the Au/S ratio. The presence of a free base, tetraphenylporphyrin (H(2)TPPOAS-), on the Au(22) core was confirmed by fluorimetric titrations with Cu(2+) and Zn(2+) ions. From all of these, the composition of the cluster was determined to be Au(22)[(-SG)(15)(-SAOPPTH(2))(2)], which was supported by mass spectrometry and elemental analysis. We utilized the fluorescence nature of these water-soluble clusters for the fabrication of fluorescent patterns by soft lithography. The patterns were studied using tapping-mode atomic force microscopy and confocal fluorescence imaging.

Do hydration dynamics follow the structural perturbation during thermal denaturation of a protein: a terahertz absorption study.

We investigate the thermal denaturation of human serum albumin and the associated solvation using terahertz (THz) spectroscopy in aqueous buffer solution. Far- and near-ultraviolet circular dichroism spectroscopy reveal that the protein undergoes a native (N) to extended (E) state transition at temperature ≤55°C with a marginal change in the secondary and tertiary structure. At 70°C, the protein transforms into an unfolded (U) state with significant irreversible disruption of its structures. We measure the concentration- and temperature-dependent THz absorption coefficient (α) of the protein solution using a p-Ge THz difference spectrometer (2.1-2.8 THz frequency range), thereby probing the collective protein-water network dynamics. When the solvated protein is heated up to 55°C and cooled down again, a reversible change in THz absorption is observed. When increasing the temperature up to 70°C, we find a dramatic irreversible change of THz absorption. The increase in THz absorption compared to bulk water is attributed to a blue shift in the spectrum of the solvated protein compared to bulk water. This is supported by measurements of THz absorption coefficients using THz time-domain spectroscopy (0.1-1.2 THz frequency range). We also use picosecond-resolved fluorescence spectroscopy of the tryptophan 214 moiety of human serum albumin. All experimental observations can be explained by a change in the hydration dynamics of the solvated protein due to the additional exposure of hydrophobic residues upon unfolding.

Modulation of Dynamics and Reactivity of Water in Reverse Micelles of Mixed Surfactants

In this contribution, we attempt to correlate the change in water dynamics in a reverse micellar (RM) core caused by the modification of the interface by mixing an anionic surfactant, sodium bis(2-ethylhexyl) sulfosuccinate (AOT), and a nonionic surfactant, tetraethylene glycol monododecyl ether (Brij-30), at different proportions, and its consequent effect on the reactivity of water, measured by monitoring the solvolysis reaction of benzoyl chloride (BzCl). The dimension of the RM droplets at different mixing ratios of AOT and Brij-30 (X(Brij-30)) has been measured using dynamic light scattering (DLS) technique. The physical properties of the RM water have been determined using Fourier transform infrared spectroscopy (FTIR) and compressibility studies, which show that with increasing X(Brij-30), the water properties tend toward that of bulk-like water. The solvation dynamics, probed by coumarin 500 dye, gets faster with X(Brij-30). The rotational anisotropy studies along with a wobbling-in-cone analysis show that the probe experiences less restriction at higher X(Brij-30). The kinetics of the water-mediated solvolysis also gets faster with X(Brij-30). The increased rate of solvolysis has been correlated with the accelerated solvation dynamics, which is another consequence of surfactant headgroup-water interaction.

Role of hydration on the functionality of a proteolytic enzyme α-chymotrypsin under crowded environment

Enzymes and other bio-macromolecules are not only sensitive to physical parameters such as pH, temperature and solute composition but also to water activity. A universally instructive way to vary water activity is the addition of osmotically active but otherwise inert solvents which also mimic the condition of an intercellular milieu. In the present contribution, the role of hydration on the functionality of a proteolytic enzyme α-chymotrypsin (CHT) is investigated by modulating the water activity with the addition of polyethylene glycols (PEG with an average molecular weight of 400). The addition of PEG increases the affinity of the enzyme to its substrate, however, followed by a decrease in the turnover number (k(cat)). Energetic calculations show that entrance path for the substrate is favoured, whereas the exit channel is restricted with increasing concentration of the crowding agent. This decrease is attributed to the thinning of the hydration shell of the enzyme due to the loss of critical water residues from the hydration surface of the enzyme as evidenced from volumetric and compressibility measurements. The overall secondary and tertiary structures of CHT determined from far-UV and near-UV circular dichroism (CD) measurements show no considerable change in the studied osmotic stress range. From kinetic and equilibrium data, we calculate 115 ± 30 numbers of water molecules to be altered during the enzymatic catalysis of CHT. Spectroscopic observation of water relaxation and rotational dynamics of ANS-CHT complex at various concentrations of the osmoting agent also support the dehydration of the hydration layer. Such dehydration/hydration processes during turnover imply a significant contribution of solvation to the energetics of the conformational changes.

Ultrafast UV-Induced Photoisomerization of Intramolecularly H-Bonded Symmetric β-Diketones

In photoinduced molecular reaction dynamics, the effects of electronic charge redistribution can lead to multiple pathways that are determined by the nature of the initial structures involved and the environment the molecule of interest is studied in. The β-diketones are a common example of this complexity. They show keto-enol tautomerism that is almost totally shifted toward the enolic form. However, compared to the gas phase, the photochemistry proceeds completely differently by virtue of the solvent environment for these compounds, which are used in commercial sunscreen agents due to a high absorption in the ultraviolet (UV) and fast deactivation processes. We disclose these dynamics by investigating three symmetrical β-diketones in various solvents. To observe these effects on an ultrafast time scale directly in the UV spectral region where the relevant electronic transitions take place, we have developed and employed femtosecond transient absorption with detection capability in the deep UV. Our studies confirm that electronic excitation of the chelated enol form does not lead to any ultrafast photochemistry other than proton transfer followed by rotamerization. The formation of the nonchelated conformers takes place on a picosecond time scale through a dark state, whereas the recovery to the stable chelated enol form is a comparably slow process.

Role of solvation dynamics in excited state proton transfer of 1-naphthol in nanoscopic water clusters formed in a hydrophobic solvent.

Excited state proton transfer (ESPT) in biologically relevant organic molecules in aqueous environments following photoexcitation is very crucial as the reorganization of polar solvents (solvation) in the locally excited (LE) state of the organic molecule plays an important role in the overall rate of the ESPT process. A clear evolution of the two photoinduced dynamics in a model ESPT probe 1‐naphthol (NpOH) upon ultrafast photoexcitation is the motive of the present study. Herein, the detailed kinetics of the ESPT reaction of NpOH in water clusters formed in hydrophobic solvent are investigated. Distinct values of time constants associated with proton transfer and solvent relaxation have been achieved through picosecond‐resolved fluorescence measurements. We have also used a model solvation probe Coumarin 500 (C500) to investigate the dynamics of solvation in the same environmental condition. The temperature dependent picosecond‐resolved measurement of ESPT of NpOH and the dynamics of solvation from C500 identify the magnitude of intermolecular hydrogen bonding energy in the water cluster associated with the ultrafast ESPT process.

Slow water dynamics at the surface of macromolecular assemblies of different morphologies

In this contribution we, for the first time, explore the slow dynamical states of confined water molecules in lamellar structures of AOT with various degrees of hydration using a picosecond resolved fluorescence spectroscopic technique using coumarin-500 as the fluorophore. A comparison of slow dynamics between AOT lamellar structures and AOT RMs have been made by preparing RMs that have a diameter the same as the interplanar water layer thickness of lamellar structures and the same number of water molecules in lamellar structures in order to understand the effect of morphology and hydration on the relaxation dynamics of water molecules in these nanoconfining systems. The relaxation time scales obtained in the lamellar systems differ to those of the RM systems and the difference of the timescales has been explained as a interplay between two opposing factors arising out of the morphology and interlayer distance, respectively. The geometrical restriction of the probe at the lamellar interface is determined by measuring time-resolved rotational anisotropy. The hydrogen bond energy of the water molecules residing at the lamellar interface is measured applying the Arrhenius type barrier crossing model.

Light driven ultrafast electron transfer in oxidative redding of Green Fluorescent Proteins

Fluorescent proteins undergoing green to red (G/R) photoconversion have proved to be potential tools for investigating dynamic processes in living cells and for photo-localization nanoscopy. However, the photochemical reaction during light induced G/R photoconversion of fluorescent proteins remains unclear. Here we report the direct observation of ultrafast time-resolved electron transfer (ET) during the photoexcitation of the fluorescent proteins EGFP and mEos2 in presence of electron acceptor, p-benzoquinone (BQ). Our results show that in the excited state, the neutral EGFP chromophore accepts electrons from an anionic electron donor, Glu222, and G/R photoconversion is facilitated by ET to nearby electron acceptors. By contrast, mEos2 fails to produce the red emitting state in the presence of BQ; ET depletes the excited state configuration en route to the red-emitting fluorophore. These results show that ultrafast ET plays a pivotal role in multiple photoconversion mechanisms and provide a method to modulate the G/R photoconversion process.

Onset of hydrogen bonded collective network of water in 1,4-dioxane.

We have studied the evolution of water hydrogen bonded collective network dynamics in mixtures of 1,4-dioxane (Dx) as the mole fraction of water (X(w)) increases from 0.005 to 0.54. The inter- and intramolecular vibrations of water have been observed using terahertz time domain spectroscopy (THz-TDS) in the frequency range 0.4-1.4 THz (13-47 cm(-1)) and Fourier transform infrared (FTIR) spectroscopy in the far-infrared (30-650 cm(-1)) and mid-infrared (3000-3700 cm(-1)) regions. These results have been correlated with the reactivity of water in these mixtures as determined by kinetic studies of the solvolysis reaction of benzoyl chloride (BzCl). Our studies show an onset of intermolecular hydrogen bonded water network dynamics beyond X(w) ≥ 0.1. At the same concentration, we observe a rapid increase of the rate constant of solvolysis of BzCl in water-Dx mixtures. Our results establish a correlation between the onset of collective hydrogen bonded network with the solvation dynamics and the activity of clustered water.