Time-dependent Fluorescence Research Articles

The Role of Imidazolium-Based Surface-Active Ionic Liquid to Restrain the Excited-State Intramolecular H-Atom Transfer Dynamics of Medicinal Pigment Curcumin: A Theoretical and Experimental Approach.

The naturally occurring polyphenolic compound curcumin has shown various medicinal and therapeutic effects. However, there are various challenges associated with curcumin, which limits its biomedical applications, such as its high degradation rate and low aqueous solubility at neutral and alkaline pH. In the present study, efforts have been directed towards trying to resolve such issues by encapsulating curcumin inside the micelles formed by imidazolium-based surface-active ionic liquid (SAIL). The shape and size of the micelles formed by the SAIL have been characterized by using DLS analysis as well as TEM measurements. The photo-physics of curcumin in the presence of ionic liquid (IL) and also with the addition of salt (NaCl) has been explored by using different optical spectroscopic tools. The time-dependent absorption studies have shown that there is relatively higher suppression in the degradation rate of curcumin after encapsulation by the imidazolium-based SAIL in an aqueous medium. The TCSPC studies have revealed that there is deactivation in the nonradiative intramolecular hydrogen transfer process of curcumin in the presence of IL micelles as well as with the addition of salt. Furthermore, the time-dependent fluorescence anisotropy measurement has been carried out to figure out the location of curcumin inside the micellar system. In order to correlate all experimental findings, density functional theory (DFT) and classical molecular dynamics (MD) simulations at neutral pH media have been performed. It has been found that the van der Waals force of interactions plays a major role in the stabilization of curcumin in the micelles rather than the coulombic forces. It also has been observed that the van der Waals interactions remain unaffected in the presence of salt. However, as revealed by the MD simulation results, the micelles are found to be more compact in size after the addition of salt. The RMSD results show that the micelles formed by the SAIL achieve greater stability after a particular time constraint. Our results have divulged that the SAIL could act as a promising drug delivery system.

Time- and reduction-dependent rise of photosystem II fluorescence during microseconds-long inductions in leaves.

Lettuce (Lactuca sativa) and benth (Nicotiana benthamiana) leaves were illuminated with 720nm background light to mix S-states and oxidize electron carriers. Green-filtered xenon flashes of different photon dose were applied and O2 evolution induced by a flash was measured. After light intensity gradient across the leaf was mathematically considered, the flash-induced PSII electron transport (= 4·O2 evolution) exponentially increased with the flash photon dose in any differential layer of the leaf optical density. This proved the absence of excitonic connectivity between PSII units. Time courses of flash light intensity and 680nm chlorophyll fluorescence emission were recorded. While with connected PSII the sigmoidal fluorescence rise has been explained by quenching of excitation in closed PSII by its open neighbors, in the absence of connectivity the sigmoidicity indicates gradual rise of the fluorescence yield of an individual closed PSII during the induction. Two phases were discerned: the specific fluorescence yield immediately increased from Fo to 1.8Fo in a PSII, whose reaction center became closed; fluorescence yield of the closed PSII was keeping time-dependent rise from 1.8Fo to about 3Fo, approaching the flash fluorescence yield Ff = 0.6Fm during 40μs. The time-dependent fluorescence rise was resolved from the quenching by 3Car triplets and related to protein conformational change. We suggest that QA reduction induces a conformational change, which by energetic or structural means closes the gate for excitation entrance into the central radical pair trap-efficiently when QB cannot accept the electron, but less efficiently when it can.

Diverse interactions of aggregated insulin with selected coumarin dyes: Time dependent fluorogenicity, simulation studies and comparison with thioflavin T

In this study, we have compared neutral coumarin based well-known commercially available probes C6, C7 and C545T for fluorogenic response from the aggregated insulin. The immediate fluorogenic responses were comparatively poor from all the three probes with respect to the previously reported response from thioflavin T (ThT) in the presence of aggregated insulin. Interestingly C6 among the three neutral coumarin derivative showed a significant steady increase of fluorescence intensity with time up to 6 h before reaching the saturation limit. Similar time dependent fluorogenic experiment with C7, C545T and ThT showed comparatively fast saturation within few minutes to 2 h. The molecular docking and simulation studies showed that these neutral probes could be stabilized in the aggregated form of the insulin predominantly by non-covalent weak interactions such as hydrogen bonding, π-π and cation-π interactions. The probability distributions of the dihedral angles between two heterocyclic parts in C6 showed maximum probability of occurrence at 0° and 180°. These probability distributions of the dihedral angles between two heterocyclic parts within all the four fluorophores provided the justification of selective time dependent fluorescence enhancement from C6 in presence of insulin aggregate. The overall fluorogenic enhancement from C6 was comparable to the fluorogenic response from ThT and theoretical study confirmed distinctly different origin of this associated slow time dependent fluorogenic response.

Variational approach to time-dependent fluorescence of a driven qubit

We employ the Dirac-Frenkel variational principle and the multiple Davydov ansatz to study time-dependent fluorescence spectra of a driven qubit in the weak to strong qubit-reservoir coupling regimes, where both the Rabi frequency and the spontaneous decay rate are comparable to the transition frequency of the qubit. Our method agrees well with the time-local master-equation approach in the weak coupling regime, and offers a flexible way to compute the spectra from the bosonic dynamics instead of two-time correlation functions. While the perturbative master equation breaks down in the strong coupling regime, our method actually becomes more accurate due to the use of bosonic coherent states under certain conditions. We show that the counter-rotating coupling between the qubit and the reservoir has considerable contributions to the photon number dynamics and the spectra under strong driving conditions even when the coupling is moderately weak. The time-dependent spectra are found to be generally asymmetric, a feature that is derived from photon number dynamics. In addition, it is shown that the spectral profiles can be dramatically different from the Mollow triplet due to strong dissipation and/or multiphoton processes associated with the strong driving. Our formalism provides a unique perspective to interpret time-dependent spectra.

Proton Transfer Dynamics in the Aprotic Proton Accepting Solvent 1-Methylimidazole.

The dynamics of proton transfer to the aprotic solvent 1-methylimidazole (MeIm, proton acceptor) from the photoacid 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) was investigated using fast fluorescence measurements. The closely related molecule, 8-methoxypyrene-1,3,6-trisulfonic acid trisodium salt (MPTS), which is not a photoacid, was also studied for comparison. Following optical excitation, the wavelength-dependent population dynamics of HPTS in MeIm resulting from the deprotonation process were collected over the entire fluorescence emission window. Analysis of the time-dependent fluorescence spectra revealed four distinct fluorescence bands that appear and decay on different time scales. We label these four states as protonated (P), associated I (AI), associated II (AII), and deprotonated (D). We find that the simple kinetic scheme of P → AI → AII → D is not consistent with the data. Instead, the kinetic scheme that describes the data has P decaying into AI, which mainly goes on to deprotonation (D), but AI can also feed into AII. AII can return to AI or decay to the ground state, but does not deprotonate within experimental error. Quantum chemistry and excited state QM/MM Born-Oppenheimer molecular dynamics simulations indicate that AI and AII are two H-bonding conformations of MeIm to the HPTS hydroxyl, axial, and equatorial, respectively.

Spectroscopic studies of the excited-state intramolecular proton and electron transfer processes of methyl benzoate derivatives in cucurbit[7]uril nanocage

The encapsulation of four methyl benzoate derivatives (I-IV) in aqueous solution in the presence of the macrocyclic host molecule cucurbit[7]uril (CB[7]) was studied using steady-state, time-resolved and 1H NMR spectroscopic techniques. The significant changes in the steady-state and time-resolved fluorescence spectra (and calculated from them fluorescence decay times) were attributed to the formation of a well-defined, stable 1:1 inclusion complex. To understand the mechanism of supramolecular solvation dynamics, the time-dependent fluorescence Stokes' shifts, represented by the normalized spectral response function c(t), were studied. Our studies have shown that solvation dynamics of water are significantly inhibited inside a cucurbit cavity. The experimental results were also used to calculate, according to Benesi-Hildebrand plot and nonlinear least-squares regression analysis, equilibrium constants of the formed inclusion complex. For all investigated dyes, the K1 value is significantly larger than this previously calculated for I-IV inclusion into cyclodextrins (CDs), indicating the superior host abilities of CB[7] as compared to CDs. The position of the guest molecule inside the host cavity was investigated by 1H NMR experiment. The nature of the 1H NMR shifts for different protons is qualitatively different for methoxy derivative-CB[7] complex (I-CB[7] and III-CB[7] systems) and hydroxy derivative-CB[7] complex (II-CB[7] and IV-CB[7] systems), which indicates that the inclusion complexes of hydroxy and methoxy derivatives with CB[7] are of a difference in nature. The CB[7]-induced chemical shifts clearly demonstrate that for II-CB[7] and IV-CB[7] complexes, benzene ring is fully embedded in the interior of CB[7], while for I-CB[7] and III-CB[7] complexes, only the part of the benzene ring is located within the CB[7] cavity. Finally, 1H NMR chemical shifts and evolution of the steady-state and time-resolved emission spectra clearly indicate that -NMe2 group attached to the III and IV is located outside of the CB[7] portal.

Anomalous Diffusion Characterization by Fourier Transform-FRAP with Patterned Illumination

Fourier transform fluorescence recovery after photobleaching (FT-FRAP) with patterned illumination is theorized and demonstrated for quantitatively evaluating normal and anomalous diffusion. Diffusion characterization is routinely performed to assess mobility in cell biology, pharmacology, and food science. Conventional FRAP is noninvasive, has low sample volume requirements, and can rapidly measure diffusion over distances of a few micrometers. However, conventional point-bleach measurements are complicated by signal-to-noise limitations, the need for precise knowledge of the photobleach beam profile, potential for bias due to sample heterogeneity, and poor compatibility with multiphoton excitation because of local heating. In FT-FRAP with patterned illumination, the time-dependent fluorescence recovery signal is concentrated to puncta in the spatial Fourier domain, with substantial improvements in signal-to-noise, mathematical simplicity, representative sampling, and multiphoton compatibility. A custom nonlinear optical beam-scanning microscope enabled patterned illumination for photobleaching through two-photon excitation. Measurements in the spatial Fourier domain removed dependence on the photobleach profile, suppressing bias from imprecise knowledge of the point spread function. For normal diffusion, the fluorescence recovery produced a simple single-exponential decay in the spatial Fourier domain, in excellent agreement with theoretical predictions. Simultaneous measurement of diffusion at multiple length scales was enabled through analysis of multiple spatial harmonics of the photobleaching pattern. Anomalous diffusion was characterized by FT-FRAP through a nonlinear fit to multiple spatial harmonics of the fluorescence recovery. Constraining the fit to describe diffusion over multiple length scales resulted in higher confidence in the recovered fitting parameters. Additionally, phase analysis in FT-FRAP was shown to inform on flow/sample translation.

Time-Resolved Probing of the Nonequilibrium Structural Solvation Dynamics by the Time-Dependent Stokes Shift.

The time-dependent fluorescence Stokes shift monitors the relaxation of the polarization of a polar solvent in the surroundings of a photoexcited solute molecule but also the structural variation of the solute following photoexcitation and the subsequent molecular charge redistribution. Here, we formulate a simple nonequilibrium quantum theory of solvation for an explicitly time-dependent continuous solvent. The time-dependent solvent induces nonequilibrium fluctuations on the solvent dynamics which are directly reflected in different time components in the time-dependent Stokes shift. We illustrate the structural dynamics in the presence of an explicitly time-dependent solvent by the example of a dynamically shrinking solute which leads to a bimodal Stokes shift. Interestingly, both contributions are mutually coupled. Furthermore, we can explain a prominent long-tail decay of the Stokes shift associated with slow structural dynamical variations.

Complete Solvation Dynamics of Coumarin 153 in Tetraalkylammonium Bromide-Based Deep Eutectic Solvents.

Deep eutectic solvents (DESs) are novel environment-friendly media for a variety of applications. In order to obtain insight into the structure and dynamics of some less-explored DESs comprising ethylene glycol and tetraalkylammonium bromide salts with variable alkyl chain length, we have captured complete dynamics occurring in these solvents in a timescale of few femtoseconds to several nanoseconds by monitoring the time-dependent fluorescence Stokes shift of coumarin 153 employing a combination of time-correlated single-photon counting and fluorescence upconversion techniques. The solvent response function constructed from the measured data reveals a sub-picosecond component (∼0.8 ps, 20-35%) in addition to a slow component (180-475 ps) with a distribution of relaxation time. The slow time component is found to be strongly dependent on the viscosity of the medium, indicating that it arises from the diffusive motions of the solvent constituents into and out of the solvation shell, whereas the ultrafast time component, which is nearly independent of the solvent viscosity, arises from fast local motions of the constituents in the immediate vicinity of the solute molecule.

Formation of CN Radical from Nitrogen and Carbon Condensation and from Photodissociation in Femtosecond Laser-Induced Plasmas: Time-Resolved FT-UV-Vis Spectroscopic Study of the Violet Emission of CN Radical.

Exploring the formation of diatomic radicals in femtosecond plasmas is important to establish the most dominant kinetic pathways following ionization and dissociation of small molecules. In this work, cyano radical formation has been studied from bromoform, acetonitrile, and methanol in nitrogen and argon plasmas created with a focused femtosecond laser beam operating at 100 kHz repetition rate and 1030 nm wavelength with 43 fs pulse length and 250 μJ pulse energy. Time-resolved Fourier transform fluorescence spectroscopy was applied in the ultraviolet-visible (UV-vis) spectral range for the characterization of the rotational and vibrational temperatures of the CN(B) radicals via fitting the experimental data. The high repetition rate of the laser allows efficient coupling with the step-scan Fourier transform spectroscopy method. Coulomb explosion at the very high intensity (∼1016 W/cm2) resulted in the formation of nascent atoms, ions, and electrons. The condensation reactions of carbon and reactive nitrogen species resulted in the formation of CN(B2Σ+) radicals and C2(d3Πg) dicarbon molecules/radicals. The CN(B) radicals were formed at the highest concentration in the case of bromoform because the weak carbon-bromine bonds resulted in reactive carbon atoms and CH radicals, which are reactive precursors for the CN(B) radical formation. In the case of acetonitrile, immediate production of CN(B) is observed with nanosecond resolution, which suggests that the CN is formed either via photodetachment or via roaming reaction associated with the Coulomb explosion of the parent molecule. The nascent rotational temperature was very high (∼6000-8500 K) and rapidly decreased in all instances within 40 ns with bromoform and acetonitrile. The highest vibrational temperature (∼7800 K) was observed in an acetonitrile/Ar mixture that decreased in about 30 ns and then increased in the observed time window. The vibrational temperature increased in all samples between 30 and 200 ns. The time dependence of fluorescence is described with a monoexponential decay in the case of acetonitrile/Ar and with biexponential decays in all other instances in the 0-250 mbar total pressure range. The shorter time constant is close to the radiative lifetime of CN(B) emission (∼60-80 ns), which can be attributed to the CN(B) radicals produced in the first few collisions at lower pressures. The longer CN(B) emission is from CN(B) created by slower chemical reactions involving carbon atoms, C2 radicals, and reactive nitrogen-containing species.

AND logic gate based fluorescence probe for simultaneous detection of peroxynitrite and hypochlorous acid.

Hypochlorous acid (HOCl) and peroxynitrite (ONOO-) are two of the most important reactive species and associated with various diseases in various physiological and pathological processes. Nonetheless, many of their roles are still vague due to the shortage of methods for simultaneously detecting HOCl and ONOO-. Herein, three simple yet useful fluorogenic probes, LG-1, LG-2 and LG-3, have been fabricated with facile synthesis route and used to monitor the coexistence of HOCl and ONOO- as AND-based logic gate fluorescent probe firstly. LG-1 and LG-2, which consists of 1,3-oxathiolane group and boronate group respectively, were designed to verify the capacity of monitoring HOCl and ONOO- without interference from each other. The result showed that these two groups are perfect reaction sites of detecting HOCl and ONOO- respectively via specific analyte-induced reactions. Hence, LG-3, which is attached by these two groups to suppress the fluorophore core, can response to HOCl and ONOO- simultaneously without mutual interference and generate the significant time-dependent fluorescence enhancement. By investigating the absorption and fluorescence properties of LG-3 towards HOCl and ONOO- individually and collectively, the result confirmed clearly that LG-3 has the capacity of monitoring the coexistence of HOCl and ONOO-, which could act as a two-input AND-based logic gate fluorescent probe.

To unravel the connection between the non-equilibrium and equilibrium solvation dynamics of tryptophan: success and failure of the linear response theory of fluorescence Stokes shift.

The connections between the non-equilibrium solvation dynamics upon optical transitions and the system's equilibrium fluctuations are explored in aqueous liquid. Linear response theory correlates time-dependent fluorescence with the equilibrium time correlation functions. In the previous work [T. Li, J. Chem. Theory Comput., 2017, 13, 1867], Stokes shift was explicitly decomposed into the contributions of various order time correlation functions on the excited state surface. Gaussian fluctuations of the solute–solvent interactions validate linear response theory. Correspondingly, the deviation of the Gaussian statistics causes the inefficiency of linear response evaluation. The above mechanism is thoroughly tested in this study. By employing molecular simulations, multiple non-equilibrium processes, not necessarily initiated from the ground state equilibrium minimum, were examined for tryptophan. Both the success and failure of linear response theory are found for this simple system and the mechanism is analyzed. These observations, assisted by the width dynamics, the initial state linear response approach, and the variation of the solvation structures, integrally verify the virtue of the excited state Gaussian statistics on the dynamics of Stokes shift.

Imidazole-coumarin containing D – A type fluorescent probe: Synthesis photophysical properties and sensing behavior for F− and CN− anion

A new class of D–A type intramolecular charge transfer (ICT) fluorescent probe, ICU has been designed and synthesized. The solvatochromic behavior, anion interaction and morphological features of the probe have been studied in detail. The molecular probe upon interaction with different class of anions showed good sensitivity for F− and CN− anions in organic medium with high detection limit and rapid response time. The time dependent fluorescence studies suggested about the dynamic and static fluorescence quenching with F− and CN− anions. The scanning electron microscopy (SEM) and diffraction light scattering (DLS) experiments have been performed to understand the morphological behavior and particle size of the probe to distinguish both anions. Upon interaction with F− and CN− the morphology of probe reveals a clear shape reconstruction from the initial aggregated spherical ball shape to fine leaf like and dumbbell like structures, respectively by disaggregation to form minimum energy structures. The photophysical and theoretical (DFT) studies all together suggested about the charge transfer process from imidazole (donor, D) to coumarin (acceptor, A) unit. The probe has been also utilized to detect the anions on test paper strips. The mechanism of interaction between probe and both the anions has been confirmed by 1HNMR and HRMS spectral data analysis.

Biomimetic Assembly of a Polydopamine Layer on Graphene as an Electron Gate for Fluorescent MicroRNA Detection in Living Cells.

A novel strategy was developed for microRNA detection based on the fluorescence quenching of polydopamine (PDA)-coated reduced graphene oxide (RGO) nanosheets (RGO@PDA). Compared with graphene oxide (GO), the reduction of GO and modification of the surface of RGO by PDA not only improve the stability, dispersity, biocompatibility, and cellular uptake without degeneration of the unique electronic properties of graphene, but also add an electron gate for harvesting electrons, as well as enabling efficient and forward electron transfer to avoid unwanted electron transfer and realize highly sensitive miRNA detection; thus a lower detection limit can be achieved in this sensing system. Remarkably, nanoprobes consisting of RGO@PDA and fluorescein-labeled single-stranded DNA can naturally enter cancer cells without the aid of transfection agents, as well as resisting enzymatic lysis and showing almost no effect on the cell viability. More importantly, intense and time-dependent fluorescence responses were observed from the important tumor marker microRNA-21 (miR-21) in living cells; thus suggesting that the proposed sensing platform shows great promise for applications in disease diagnosis and fundamental research into biochemistry.

Hybrid equilibrium-nonequilibrium molecular dynamics approach for two-dimensional solute-pump/solvent-probe spectroscopy.

There has been a tremendous effort in accessing liquid-phase solvation dynamics using ultrafast spectroscopies for decades. It is recently demonstrated that one can go beyond tracking the relaxation of the solute-solvent interaction energy as reported in traditional time-dependent fluorescence spectroscopy. Combining a resonant solute pump with a subsequent nonresonant 4-wave-mixing light-scattering solvent probe gives rise to two-dimensional solute-pump/solvent probe (SPSP) spectroscopy, which displays the nonequilibrium relaxation of the intermolecular vibrations after the solute gets electronically excited. However, the response function of the SPSP spectra has been challenging to calculate, even on the classical-mechanical level, due to the difficulty of evaluating the surviving Poisson bracket. In this work, a hybrid equilibrium-nonequilibrium molecular dynamics simulation approach is proposed where the Poisson bracket can be estimated using nonequilibrium molecular dynamics. Applying the resulting numerically exact formalism and the previously proposed hybrid instantaneous-normal-mode/molecular-dynamics approach to a preferential solvation model system reveals that the SPSP spectroscopy provides an alternative measure for solvation dynamics, which is more sensitive to the local solvent structures than the traditional energetic dynamics measured in the time-dependent fluorescence spectroscopy.

Synthesis of Strongly Fluorescent Imidazole Derivatives: Structure Property Studies, Halochromism and Fluorescent Photoswitching.

New series of methoxy and hydroxyl group substituted triphenylamine (TPA)-imidazole fluorescent molecules (5-(diphenylamino)-2-(1H-phenanthro[9,10-d]imidazol-2-yl)phenol (1), 5-(diphenylamino)-2-(1-phenyl-1H-phenanthro[9,10-d]imidazol-2-yl)phenol (2), 5-(diphenylamino)-2-(4,5-diphenyl-1H-imidazol-2-yl)phenol (3), 5-(diphenylamino)-2-(1,4,5-triphenyl-1H-imidazol-2-yl)phenol (4), N-(3-methoxy-4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenyl)-N-phenylbenzenamine (5), N-(3-methoxy-4-(1-phenyl-1H-phenanthro[9,10-d]imidazol-2-yl)phenyl)-N-phenylbenzene amine (6), and N-(3-methoxy-4-(4,5-diphenyl-1H-imidazol-2-yl)phenyl)-N-phenylbenzenamine (7)) have been synthesized that exhibited strong solution fluorescence and molecular structure and conformation controlled fluorescence photoswitching, solid state fluorescence and halochromism. Hydroxyl substituted molecules (1-4) showed moderate to strong fluorescence in solution depend on solvent polarity and very weak solid state fluorescence. Methoxy substituted molecules (5-7) displayed strong fluorescence both in solution and solid state. Solid state structural studies revealed strong intramolecular H-bonding in the crystal lattice. Interestingly, highly twisted structure (6) showed rare light induced reversible fluorescence switching in CHCl3. The observation of isobestic point in time dependent fluorescence photoswitching studies indicated structural isomer conversion. Further, acid sensitive imidazole nitrogen has been made use to demonstrate solid state fluorescence switching via halochromism. Thus the present studies attempted to develop new fluorescent molecules and establish structure-property relationship for designing fluorescence switching materials. Graphical Abstract Molecular structure controlled solid state fluorescence, halochromism and a rare fluorescence photoswitching in chloroform have been observed with triphenylamine-imidazole derivatives.

Binding of lanthanide salts to zwitterionic phospholipid micelles

As the use of lanthanide salts in biophysical systems increases and the separation of lanthanides from nuclear and other wastes with extraction processes has become an important technological challenge, a deeper understanding of the behavior of lanthanides at lipid interfaces is urgently needed. In this work the interaction of lanthanide salts with zwitterionic phospholipids is probed using aqueous micelles of the surfactant dodecyl phosphocholine (DPC), which are useful membrane-mimetic model systems, widely used for the solubilization of membrane proteins in aqueous solutions. Because more than one species exists in lanthanide salt solutions, even at the pH value of 4 used in this experiment, the major goal of this investigation is to examine which species are actually binding to the micelles. Using static and time-dependent europium fluorescence, strong indications are obtained that both the Eu3+ cation and its 1:1 chloride, nitrate, or sulfate complexes bind to the micelles, whereas the europium species do not appear to interact strongly with DPC molecules below the cmc. From isothermal titration calorimetry (ITC) measurements it is found that the lanthanide interaction with DPC micelles increases to the right of the lanthanide series and is - surprisingly – endothermic, underlying the important role of hydration effects in the interaction. The anion of the lanthanide salt strongly influences the thermodynamics: perchlorate and sulfate salts give extraordinary results, switching the interaction to exothermic. A multi-level phenomenological electrostatic model of the europium fluorescence lifetimes strongly suggests that in the case of nitrate salts both Ln3+ and LnNO32+ ions bind to the micelles. Overall a detailed molecular picture of the complexity of lanthanide-lipid interactions at interfaces is emerging from these experiments and the associated modelling effort.

Modulation of Kinetic Pathways of Enzyme-Substrate Interaction in a Microfluidic Channel: Nanoscopic Water Dynamics as a Switch.

Enzyme-mediated catalysis is attributed to enzyme-substrate interactions, with models such as "induced fit" and "conformational selection" emphasizing the role of protein conformational transitions. The dynamic nature of the protein structure, thus, plays a crucial role in molecular recognition and substrate binding. As large-scale protein motions are coupled to water motions, hydration dynamics play a key role in protein dynamics, and hence, in enzyme catalysis. Here, microfluidic techniques and time-dependent fluorescence Stokes shift (TDFSS) measurements are employed to elucidate the role of nanoscopic water dynamics in the interaction of an enzyme, α-Chymotrypsin (CHT), with a substrate, Ala-Ala-Phe-7-amido-4-methylcoumarin (AMC) in the cationic reverse micelles of benzylhexadecyldimethylammonium chloride (BHDC/benzene) and anionic reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT/benzene). The kinetic pathways unraveled from the microfluidic setup are consistent with the "conformational selection" fit for the interaction of CHT with AMC in the cationic reverse micelles, whereas an "induced fit" mechanism is indicated for the anionic reverse micelles. In the cationic reverse micelles of BHDC, faster hydration dynamics (≈550 ps) aid the pathway of "conformational selection", whereas in the anionic reverse micelles of AOT, the significantly slower dynamics of hydration (≈1600 ps) facilitate an "induced fit" mechanism for the formation of the final enzyme-substrate complex. The role of water dynamics in dictating the mechanism of enzyme-substrate interaction becomes further manifest in the neutral reverse micelles of Brij-30 and Triton X-100. In the former, the faster water dynamics aid the "conformational selection" pathway, whereas the significantly slower dynamics of water molecules in the latter are conducive to the "induced fit" mechanism in the enzyme-substrate interaction. Thus, nanoscopic water dynamics act as a switch in modulating the pathway of recognition of an enzyme (CHT) by the substrate (AMC) in reverse micelles.

Optical and impedimetric study of genetically modified cells for diclofenac sensing

Abstract. Whole-cell biosensors, based on genetically modified yeast cells, were employed to detect anthropogenic micropollutants (e.g. drugs). Specific stimuli, e.g. traces of drugs, lead to the induction of fluorescence in the respective cells. Receptors of the cells detect specific signal molecules and induce the formation of fluorescent proteins. In this work, genetically modified cells of the yeast Saccharomyces cerevisiae BY4741 were confined in a four-chamber microfluidic cell, providing an optical monitoring of the cell behaviour and their supply with the nutrients. The measurements of the time-dependent fluorescence intensity were performed with different concentrations of the drug diclofenac, and the sensitivity of yeast cells to diclofenac was demonstrated. Cell viability was monitored by simultaneous impedance recording.

(Invited) Distinct Coating Displacement Kinetics for Single-Wall Nanotube Enantiomers

It is known that specific oligomers of single-stranded DNA (ssDNA) can show selectivity toward different (n,m) structural species of single-wall carbon nanotubes (SWCNTs). These selective interactions can also extend to the left- and right-handed helical forms of a single (n,m) species. An example of this is the ability of the (ATT)4 oligo to strongly distinguish between the two optical isomers of (7,5) SWCNTs. We show that this helicity-selective coating interaction causes a resolvable shift between the two enantiomers’ fluorescence spectra. Most strikingly, the room temperature rates of displacement of the (ATT)4 coating on exposure to sodium deoxycholate are found to differ by two orders of magnitude. This is shown from measurements of time dependent fluorescence spectra. During coating displacement, the enantiomer with high affinity for the ssDNA oligomer is deduced to form an intermediate hybrid that is not observed for the low affinity enantiomer. These results demonstrate the diverse and dramatic enantiomeric differences in properties of SWCNTs complexed with ssDNA.