Competition among recombination pathways in single FAPbBr3 nanocrystals.

Single particle level microscopy of immobilized FAPbBr3 nanocrystals (NCs) has elucidated the involvement of different processes in their photoluminescence (PL) intermittency. Four different blinking patterns are observed in the data from more than 100 NCs. The dependence of PL decays on PL intensities brought out in fluorescence lifetime intensity distribution (FLID) plots is rationalized by the interplay of exciton- and trion-mediated recombinations along with hot carrier (HC) trapping. The high intensity-long lifetime component is attributed to neutral exciton recombination, the low intensity-short lifetime component is attributed to trion assisted recombination, and the low intensity-long lifetime component is attributed to hot carrier recombination. Change-point analysis (CPA) of the PL blinking data reveals the involvement of multiple intermediate states. Truncated power law distribution is found to be more appropriate than power law and lognormal distribution for on and off events. Probability distributions of PL trajectories of single NCs are obtained for two different excitation fluences and wavelengths (λex = 400, 440 nm). Trapping rate (kT) prevails at higher power densities for both excitation wavelengths. From a careful analysis of the FLID and probability distributions, it is concluded that there is competition between the HC and trion assisted blinking pathways and that the contribution of these mechanisms varies with excitation wavelength as well as fluence.

Does Viscosity Decoupling Guarantee Dynamic Heterogeneity? A Clue through an Excitation and Emission Wavelength-Dependent Time-Resolved Fluorescence Anisotropy Study.

Traditionally, deviation from Stokes-Einstein-Debye (SED) relation in terms of viscosity dependence of medium dynamics, i.e., τx∝(ηT)p with p ≠ 1, is taken as a signature of dynamic heterogeneity. However, it does not guarantee medium heterogeneity, as the decoupling may also originate from the deviation of the basic assumption of SED. Here, we developed a method to find a stronger relation between viscosity decoupling (p ≠ 1) and dynamic heterogeneity in terms of rotational motion. Our approach exploited the fact that in heterogeneous media, a solvatochromic probe will be solvated to a different extent at different microdomains (subpopulations), and photoselection of these subpopulations can be achieved by excitation or emission wavelength-dependent measurements. We hypothesized that the dynamics of a homogeneous system might show viscosity decoupling, but the extent of decoupling at different excitations (or at different emissions) should not be different. On the other hand, in a heterogeneous medium, this extent of viscosity decoupling (p-value) should be different at different excitations (or at different emissions). As proof of concept, we investigated three versatile solvent media: squalane (viscous molecular liquid), 1-ethyle-3-methylimidazolium ethyl sulfate ionic liquid (IL), and [0.78 acetamide + 0.22 LiNO3] deep eutectic solvent (DES). We found that squalane is homogeneous, although it shows fractional viscosity dependence (p ≠ 1). Interestingly, mild heterogeneity in IL and significant heterogeneity in the DES were observed. Overall, we conclude that the difference in the p-value as a function of excitation (or emission) wavelength-dependent might be a superior way for the detection of dynamic heterogeneity.

Associated Water Dynamics Might Be a Key Factor Affecting Protein Stability in the Crowded Milieu.

Over the past 20 years, the most studied and debated aspect of macromolecular crowding is how it affects protein stability. Traditionally, it is explained by a delicate balance between the stabilizing entropic effect and the stabilizing or destabilizing enthalpic effect. However, this traditional crowding theory cannot explain experimental observations like (i) negative entropic effect and (ii) entropy-enthalpy compensation. Herein, we provide experimental evidence that associated water dynamics plays a crucial role in controlling protein stability in the crowded milieu for the first time. We have correlated the modulation of associated water dynamics with the overall stability and its individual components. We showed that rigid associated water would stabilize the protein through entropy but destabilize it through enthalpy. In contrast, flexible associated water destabilizes the protein through entropy but stabilizes through enthalpy. Consideration of entropic and enthalpic modulation through crowder-induced distortion of associated water successfully explains the negative entropic part and entropy-enthalpy compensation. Furthermore, we argued that the relationship between the associated water structure and protein stability should be better understood by individual entropic and enthalpic components instead of the overall stability. Although a huge effort is necessary to generalize the mechanism, this report provides a unique way of understanding the relationship between protein stability and associated water dynamics, which might be a generic phenomenon and should trigger much research in this area.

Does Microsecond Active-Site Dynamics Primarily control Proteolytic Activity of Bromelain? Clues from Single Molecular Level Study with a Denaturant, a Stabilizer and a Macromolecular Crowder.

Proteins are dynamic entity with various molecular motions at different timescale and length scale. Molecular motions are crucial for the optimal function of an enzyme. It seems intuitive that these motions are crucial for optimal enzyme activity. However, it is not easy to directly correlate an enzyme's dynamics and activity due to biosystems' enormous complexity. amongst many factors, structure and dynamics are two prime aspects that combinedly control the activity. Therefore, having a direct correlation between protein dynamics and activity is not straightforward. Herein, we observed and correlated the structural, functional, and dynamical responses of an industrially crucial proteolytic enzyme, bromelain with three versatile classes of chemicals: GnHCl (protein denaturant), sucrose (protein stabilizer), and Ficoll-70 (macromolecular crowder). The only free cysteine (Cys-25 at the active-site) of bromelain has been tagged with a cysteine-specific dye to unveil the structural and dynamical changes through various spectroscopic studies both at bulk and at the single molecular level. Proteolytic activity is carried out using casein as the substrate. GnHCl and sucrose shows remarkable structure-dynamics-activity relationships. Interestingly, with Ficoll-70, structure and activity are not correlated. However, microsecond dynamics and activity are beautifully correlated in this case also. Overall, our result demonstrates that bromelain dynamics in the microsecond timescale around the active-site is probably a key factor in controlling its proteolytic activity.

Temperature-Dependent Dielectric Relaxation in Ionic Acetamide Deep Eutectics: Partial Viscosity Decoupling and Explanations from the Simulated Single-Particle Reorientation Dynamics and Hydrogen-Bond Fluctuations.

We report here temperature-dependent (293 ≤ T (K) ≤ 336) dielectric relaxation (DR) measurements of (acetamide + LiBr/NO3-/ClO4-) deep eutectic solvents (DESs) in the frequency window of 0.2 ≤ ν (GHz) ≤ 50 and explore, via molecular dynamics simulations, the relative roles for the collective single-particle reorientational relaxations and the H-bond dynamics of acetamide in the measured DR response. In addition, DR measurements of neat molten acetamide were performed. Recorded DR spectra of these DESs require multi-Debye fits and produce well-separated DR time scales that are spread over several picoseconds to ∼1 ns. Simulations suggest DR time scales derive contributions from both the collective reorientational (Cl(t)) relaxation and structural H-bond (CHB(t)) dynamics of acetamide. A good correlation between the measured and simulated activation energies further reveals a strong connection between the measured DR and the simulated Cl(t) and CHB(t). Average DR times exhibit a strong fractional viscosity dependence, suggesting substantial microheterogeneity in these media. Simulations of Cl(t) and CHB(t) reveal strong stretched exponential relaxations with a stretching exponent, 0.4 ≤ ß ≤ 0.7. The ratio between the average reorientational correlation times of first and second ranks, ⟨τ⟩l=1/⟨τ⟩l=2, deviates appreciably from Debye's l(l+1) law for homogeneous media. Importantly, a pronounced translation-rotation decoupling between the simulated reorientation and center-of-mass diffusion times was observed.

Subpicosecond Solvation Response and Partial Viscosity Decoupling of Solute Diffusion in Ionic Acetamide Deep Eutectic Solvents: Fluorescence Up-Conversion and Fluorescence Correlation Spectroscopic Measurements.

Fluorescence up-conversion (∼250 fs instrumental response) coupled with time correlated single photon counting measurements was performed to explore the complete Stokes shift dynamics of a dipolar solute probe, coumarin 153 (C153), in several ionic acetamide deep eutectic solvents (DESs) that contained lithium nitrate/bromide/perchlorate as electrolyte. Combined measurements near room temperature reflected a total dynamic Stokes shift of approximately 800-1100 cm-1 and triexponential solvation response functions. Interestingly, the average rate of solvation became faster upon successive replacement of bromide by nitrate in these deep eutectics, and a subpicosecond time scale emerged in the measured solvation response when bromide was fully replaced by nitrate. Temperature dependent solute diffusion in these deep eutectics at the single molecule level, monitored by tracking the translational motion of rhodamine 6G (R6G) via fluorescence correlation spectroscopic (FCS) technique, revealed pronounced fractional viscosity dependence of the solute's translational motion. Subsequently, this partial decoupling of solute translation was attributed to the microheterogeneous nature of these ionic DESs after examining the diffusion-viscosity relationship via the FCS measurements of R6G in several normal solvents at room temperature and in a liquid amide solvent at different temperatures.

Dynamics at the non-ionic micelle/water interface: Impact of linkage substitution.

The impact of atom substitution on the glycoside linkage bridging the head and the tail parts in a nonionic surfactant molecule on aqueous dynamics of the resultant micellar solutions has been explored, employing time-resolved fluorescence and dielectric relaxation (DR) measurements. We have utilized n-octyl-ß-D-glucopyranoside (OG) and n-octyl-ß-D-thioglucopyranoside (OTG) as nonionic surfactants where the oxygen atom in the glucopyranoside unit is substituted by a sulfur atom. The substitution impact is immediately reflected in the dynamic light scattering measurements of aqueous solutions where the estimated size of the OTG micelles is found to be approximately four times larger than the OG micelles. Steady state spectral features obtained by using a fluorescent probe solute, coumarin 153 (C153), in these micellar solutions are quite similar and indicate locations of the solute at the micelle/water interface for both the surfactants. Interestingly, significant differences in the rotational and solvation dynamics of C153 in these two micellar solutions have been registered. The corresponding DR measurements do not indicate any signature of relaxation typical of bound water. The absence of bound water is further supported by the differential scanning calorimetric measurements. However, the typical slow solvation time scale for aqueous micellar solutions has been observed for these surfactants. Fluctuations in the solute-interface interaction energy due to the solute motion has been argued to be the origin for this slow solvation component as DR measurements do not indicate the presence of qualitatively similar relaxation time scale in the medium.

Interaction and Dynamics in a Fully Biodegradable Glucose-Containing Naturally Abundant Deep Eutectic Solvent: Temperature-Dependent Time-Resolved Fluorescence Measurements.

A new room-temperature deep eutectic solvent (DES) composed of glucose, urea, and water has been prepared and its relaxation dynamics explored via temperature-dependent time-resolved fluorescence measurements employing hydrophilic and hydrophobic solute probes. Differential scanning calorimetry measurements indicate a glass transition temperature (Tg) of ∼236 K. Measured viscosity coefficients (η) vary from ∼600 to ∼100 cP in the temperature range 318 ≤ T/K ≤ 343 and exhibit Arrhenius-type temperature dependence with an activation energy of ∼65 kJ mol-1. Interestingly, this DES forms a stable liquid at ∼300 K but is too viscous to be accurately measured by us below 318 K. Temperature-dependent dynamic fluorescence anisotropy measurements using hydrophobic and hydrophilic solutes of similar sizes reveal bi-exponential kinetics and Arrhenius-type temperature dependence for solute rotation times (⟨τr⟩) but with significantly decreased activation energies, ∼31 kJ mol-1 (hydrophobic) and ∼21 kJ mol-1 (hydrophilic). Deviation from hydrodynamics is further reflected in the strong fractional viscosity dependence of ⟨τr⟩: ⟨τr⟩ ∝ (η/T)p with p ≈ 0.3-0.5, indicating pronounced temporal heterogeneity in the relaxation dynamics. Dynamic fluorescence Stokes shift measurements (temporal resolution ∼85 ps) produce dynamic shifts of ∼500-700 cm-1, bi-exponential solvation energy relaxation with time constants in the range ∼0.2 ns and ∼4 ns, and estimated missing amplitudes of ∼65-75%. Impact of the density difference between a nonpolar solvent and this DES on the estimated missing amplitudes is explored via measuring the temperature-dependent densities and refractive indices of this DES. Lifetime measurements suggest considerable temperature dependence for the hydrophobic solute but no such dependence for the hydrophilic one. Excitation energy dependence of fluorescence emission of various solutes with widely different lifetimes indicates mild spatial heterogeneity for this DES.

Exploring Aqueous Solution Dynamics of an Amphiphilic Diblock Copolymer: Dielectric Relaxation and Time-Resolved Fluorescence Measurements.

We explore in this work, after synthesizing and appropriately characterizing an amphiphilic diblock copolymer, its interaction with water molecules and the subsequent aqueous solution dynamics by employing time-resolved fluorescence measurements (TRF) and megahertz-gigahertz dielectric relaxation (DR) experiments. The synthesized amphiphilic diblock copolymer is poly(2-(((tert-butoxycarbonyl)alanyl)oxy)ethyl methacrylate)-b-poly(polyethylene glycol monomethyl ether methacrylate) (P(Boc-l-Ala-HEMA)-b-PPEGMA). Dynamic light scattering measurements of aqueous solutions indicate formation of 14-20 nm particles from a balance between the chain lengths of the hydrophobic (P(Boc-l-Ala-HEMA) and hydrophilic (PPEGMA) segments. Field-emission scanning electron microscopy, on the other hand, suggests a spherical shape for the dried micelles. The critical micelle concentration of the P(Boc-l-Ala-HEMA)-b-PPEGMA block copolymer at different block lengths in aqueous media, determined via steady-state fluorescence measurements, is very low (∼4-8 mg/L), and the resultant micellar size has been found to be insensitive to the polymer concentration. Interfacial and bulk aqueous dynamics have been investigated by tracking the solution frictional resistance on rotational motion of dissolved hydrophobic and hydrophilic dipolar solute probes of comparable sizes. TRF anisotropy measurements reflect the biphasic temporal profile for the frictional resistance. Interestingly, the hydrophobic probe, because of its preferential location at the micellar interface, experiences greater frictional resistance than the hydrophilic counterpart, although the latter reports stronger polymer concentration dependence of the frictional retardation than the former. DR measurements at the highest of the polymer concentrations considered suggest presence of aqueous dynamics slower than that for neat bulk water, although evidence for such "slow" dynamics at lower concentrations has not been detected in the present DR measurements.

Dielectric relaxation in acetamide + urea deep eutectics and neat molten urea: Origin of time scales via temperature dependent measurements and computer simulations.

Dielectric relaxation (DR) measurements in the frequency window 0.2 ≤ ν(GHz) ≤ 50 for deep eutectic solvents (DESs) made of acetamide (CH3CONH2) and urea (NH2CONH2) with the general composition, [f CH3CONH2 + (1 - f) NH2CONH2] at f = 0.6 and 0.7, reveal three distinct relaxation time scales-τ1 ∼ 120 ps, τ2 ∼ 40 ps, and τ3 ∼ 5 ps. Qualitatively similar time scales have been observed for DR of neat molten urea, whereas the reported DR for neat molten acetamide in the same frequency window reflects two relaxation processes with no trace of ∼100 ps time scale. This slowest DR time scale (τ1) resembles closely to the long-time constant of the simulated structural H-bond relaxation (CHB(t)) involving urea pairs. Similarity in activation energies estimated from the temperature dependent DR measurements (335 ≤ T/K ≤ 363) and structural H-bond relaxations indicates that the structural H-bond relaxation overwhelmingly dominates the slowest DR relaxation in these DESs. Simulated collective reorientational correlation functions (C ℓ (t)), on the other hand, suggest that the second slower time scale (∼40 ps) derives contributions from both the single particle orientation dynamics and structural H-bond relaxation, leaving no role for hydrodynamic molecular rotations. The sub-10 ps DR time scale has been found to be connected to the fast reorientation dynamics of the component molecules (acetamide or urea). Fractional viscosity dependence for the longest DR times, τ DR ∝ η / T p , has been observed for these DESs with the fraction power p = 0.7. Subsequently, the temporal heterogeneity aspects of these media have been investigated by examining the simulated particle motion characteristics and substantiated by estimating the dynamically correlated time scales and length-scales through simulations of four-point susceptibilities and density correlations. These estimated dynamical time scales and length-scales assist in explaining the different inferences regarding solution heterogeneity drawn from different measurements on these DESs.