Tridib Mondal

Diffusion of Organic Dyes in Ionic Liquid and Giant Micron Sized Ionic Liquid Mixed Micelle: Fluorescence Correlation Spectroscopy

Diffusion of organic dyes in neat room temperature ionic liquid (RTIL) and RTIL-mixed micelle has been studied by fluorescence correlation spectroscopy (FCS). We have selected two RTILs, 3-pentyl-1-methyl imidazolium bromide ([C5C1Im][Br]) and the corresponding tetra-fluoroborate ([C5C1Im][BF(4)]). Diffusion coefficients (D(t)) of three organic dyes--DCM (neutral), C480 (neutral), and C343 (anionic)--in these RTILs are ∼100 times slower compared to water. This indicates very high viscosity of the RTILs. In contrast to water, the D(t) in RTIL exhibits a wide distribution which suggests the presence of heterogeneity (nanoscale organization). The presence of ions in the RTILs markedly affects diffusion in the RTILs. D(t)s of C480 (neutral) and C343 (anionic) are very similar in water but in RTILs the ionic dye C343 diffuses 1.7 times slower than neutral C480. This is attributed to the electrostatic force exerted by the ions in the RTILs. In the giant (∼2-4 μm) [C5C1Im][Br]-triblock copolymer (P123) mixed micelle D(t) of DCM, C480, and C343 are found to be 7, 15, and 7 μm(2) s(-1), respectively. The results are compared with those in P123 micelle and gel.

An FCS study of unfolding and refolding of CPM-labeled human serum albumin: role of ionic liquid.

The effect of a room temperature ionic liquid (RTIL) on the conformational dynamics of a protein, human serum albumin (HSA), is studied by fluorescence correlation spectroscopy (FCS). For this, the protein was covalently labeled by a fluorophore, 7-dimethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). On addition of a RTIL ([pmim][Br]) to the native protein, the diffusion coefficient (D(t)) decreases and the hydrodynamic radius (R(h)) increases. This suggests that the RTIL ([pmim][Br]) acts as a denaturant when the protein is in the native state. However, addition of [pmim][Br] to a protein denatured by GdnHCl causes an increases in D(t) and decrease in R(h). This suggests that in the presence of GdnHCl addition of RTIL helps the protein to refold. In the native state, the conformational dynamics of protein is described by three distinct time constants: ~3.6 ± 0.7, ~29 ± 4.5, and 133 ± 23 μs. The faster components (~3.6 ± 0.7 and ~29 ± 4.5 μs) are ascribed to chain dynamics of the protein, while the slowest component (133 μs) is responsible for interchain interaction or concerted motion. On addition of [pmim][Br], the conformational dynamics of HSA becomes slower (~5.1 ± 1, ~43.5 ± 2.8, and ~311 ± 2.3 μs in the presence of 1.5 M [pmim][Br]). The time constants for the protein denatured by 6 M GdnHCl are 3.2 ± 0.4, 34 ± 6, and 207 ± 38 μs. When 1.5 M [pmim][Br] is added to the denatured protein (in 6 M GdnHCl), the time constants become ~5 ± 1, ~41 ± 10, and ~230 ± 45 μs. The lifetime histogram shows that, on addition of GdnHCl to HSA, the contribution of the shorter lifetime component decreases and vanishes at 6 M GdnHCl. The shorter lifetime component immediately reappears after addition of RTIL to unfolded HSA. This suggests recoiling of the unfolded protein by RTIL.

Ultrafast and ultraslow proton transfer of pyranine in an ionic liquid microemulsion.

Effect of a room temperature ionic liquid (RTIL) and water on the ultrafast excited state proton transfer (ESPT) of pyranine (8-hydroxypyrene-1,3,6-trisulfonate, HPTS) inside a microemulsion is studied by femtosecond up-conversion. The microemulsion consists of the surfactant, triton X-100 (TX-100) in benzene (bz) and contains the RTIL, 1-pentyl-3-methyl-imidazolium tetrafluoroborate ([pmim] [BF(4)]) as the polar phase. In the absence of water, HPTS undergoes ultrafast ESPT inside the RTIL microemulsion (RTIL/TX-100/bz) and the deprotonated form (RO(-)) exhibits three rise components of 0.3, 14, and 375 ps. It is proposed that in the RTIL microemulsion, HPTS binds to the TX-100 at the interface region and participates in ultrafast ESPT to the oxygen atoms of TX-100. On addition of water an additional slow rise of 2150 ps is observed. Similar long rise component is also observed in water/TX-100/benzene reverse micelle (in the absence of [pmim] [BF(4)]). It is suggested that the added water molecules preferentially concentrate (trapped) around the palisade layer of the RTIL microemulsion. The trapped water molecules remain far from the HPTS both in the presence and absence of ionic liquid and gives rise to the slow component (2150 ps) of ESPT. Replacement of H(2)O by D(2)O causes an increase in the time constant of the ultraslow rise to 2350 ps.

Excited state proton transfer in ionic liquid mixed micelles.

Excited state proton transfer (ESPT) of pyranine (8-hydroxypyranine-1,3,6-trisulfonate, HPTS) in room temperature ionic liquid (RTIL) mixed micelles is studied by femtosecond up-conversion. The mixed micelle consists of a triblock copolymer, (PEO)(20)-(PPO)(70)-(PEO)(20) (Pluronic P123), and one of the two RTILs, 1-pentyl-3-methyl-imidazolium bromide ([pmim][Br]) and 1-pentyl-3-methyl-imidazolium tetra-fluoroborate ([pmim][BF(4)]). The size and structure of the mixed micelle vary with the relative amount of the RTIL. For [pmim][Br], the hydrodynamic diameter of the mixed micelle is 26 nm in 0.3 M RTIL and 3500 nm in 3.0 M RTIL. The time constant of initial proton transfer (τ(PT)) in P123 micelle (65 ps) is 10 times slower than that (5 ps) in water, while the time constants of recombination (τ(rec)) and dissociation (τ(diss)) are 2-3 times slower in P123 micelle. On addition of the RTIL, the rate of ESPT is markedly modified. In 0.3 M RTIL-P123 mixed micelle, τ(PT) is shorter than that in P123 micelle. In the mixed micelle, τ(PT) increases with an increase in the concentration of the RTIL (230 ps in 3 M [pmim][Br] and 55 ps in 0.9 M [pmim][BF(4)]). This is attributed to large scale penetration of the P123 micelle by RTIL replacing water molecules. The time constants of proton transfer (τ(PT), τ(rec), and τ(diss)) are faster than the slowest component (200-500 ps) of solvation dynamics. It seems that the ultrafast component of solvation (<0.3 ps and <5 ps) is enough for inducing proton transfer. The time constant of the proton transfer (τ(PT)) in [pmim][BF(4)]-P123 mixed micelle is longer (∼20%) than that in [pmim][Br]-P123 mixed micelle for the same concentration of RTIL. The counterion dependence of ESPT is attributed to the difference in the structure and greater hydrophobicity of the [pmim][BF(4)].

Dynamics in Cytoplasm, Nucleus, and Lipid Droplet of a Live CHO Cell: Time-Resolved Confocal Microscopy

Different regions of a single live Chinese hamster ovary (CHO) cell are probed by time-resolved confocal microscopy. We used coumarin 153 (C153) as a probe. The dye localizes in the cytoplasm, nucleus, and lipid droplets, as is clearly revealed by the image. The fluorescence correlation spectroscopy (FCS) data shows that the microviscosity of lipid droplets is ~34 ± 3 cP. The microviscosities of nucleus and cytoplasm are found to be 13 ± 1 and 14.5 ± 1 cP, respectively. The average solvation time () in the lipid droplets (3600 ± 50 ps) is slower than that in the nucleus ( = 750 ± 50 ps) and cytoplasm ( = 1100 ± 50 ps). From the position of emission maxima of C153, the polarity of the nucleus is estimated to be similar to that of a mixture containing 26% DMSO in triacetin (η ~ 11.2 cP, ε ~ 26.2). The cytoplasm resembles a mixture of 18% DMSO in triacetin (η ∼ 12.6 cP, ε ∼ 21.9). The polarity of lipid droplets is less than that of pure triacetin (η ~ 21.7 cP, ε ~ 7.11).

Ultrafast FRET in Ionic Liquid-P123 Mixed Micelles: Region and Counterion Dependence

Ultrafast fluorescence resonance energy transfer (FRET) in a mixed micelle containing a room-temperature ionic liquid (RTIL) is studied by picosecond and femtosecond emission spectroscopy. The mixed micelle consists of a triblock copolymer, (PEO)(20)-(PPO)(70)-(PEO)(20) (Pluronic P123), and a RTIL, 1-pentyl-3-methyl-imidazolium tetra-flouroborate, ([pmim][BF(4)]) or 1-pentyl-3-methyl-imidazolium bromide ([pmim][Br]). Coumarin 480 (C480) is used as the donor, and the acceptor is rhodamine 6G (R6G). Multiple time scales of FRET were detected-an ultrashort component of 1-3 ps and two relatively long components (300-400 ps and 2500-3500 ps). The different time scales are attributed to different donor-acceptor distances. It is proposed that the ionic acceptor (R6G) is localized in the polar corona region of the mixed micelle, while the neutral donor (C480) is distributed over both corona and hydrophobic core regions. The ultrafast (1-3 ps) components are assigned to FRET at a close contact of donor and acceptor. This occurs for the donor in the polar corona region in close proximity of the acceptor. The longer components (300-400 ps and 2500-3500 ps) arise from long-distance FRET from the donor at the core and the acceptor at the corona region. The relative contribution of the ultrafast component of FRET (∼3 ps) increases from 5% at λ(ex) = 375 nm to 30% at λ(ex) = 435 nm in the 0.3 M [pmim][BF(4)] mixed micelle and from 25 to 100% in the 0.9 M [pmim][BF(4)] mixed micelle. It is suggested that, at λ(ex) = 435 nm, mainly the donor molecules present at the corona are excited, causing ultrafast FRET due to a short donor-acceptor distance. At shorter λ(ex), the donor (C480) molecule at the core regions is excited, giving rise to a very long 3400 ps component (R(DA) ∼ 50 Å). Thus, λ(ex) variation leads to excellent spatial resolution. The counterion dependence (Br(-) vs BF(4)(-)) is attributed to the difference in the local polarity and size of the two mixed micelles.

Salt effect on the ultrafast proton transfer in niosome.

Excited state proton transfer (ESPT) of pyranine (8-hydroxypyranine-1,3,6-trisulfonate, HPTS) in a niosome is studied by fluorescence correlation spectroscopy (FCS) and femtosecond up-conversion. The niosome consists of a neutral surfactant triton X-100 (TX-100) and cholesterol. FCS studies suggest that in the presence of niosome almost all of the HPTS is transferred to the niosome and the amount of free HPTS present in bulk water is negligible. The time constant of initial proton transfer (τ(PT)) in niosome (40 ps) is ∼8 times slower than that (5 ps) in bulk water, while the time constants of recombination (τ(rec)) and dissociation (τ(diss)) are ∼4 times and ∼1.5 times slower in niosome, respectively. On addition of NaCl, the rate of ESPT is markedly retarded both in free water and in niosome. In the niosome, τ(PT) slows down to 80 ps in 1 M NaCl and 225 ps in 4 M NaCl.

Marcus-like inversion in electron transfer in neat ionic liquid and ionic liquid-mixed micelles.

Ultrafast photoinduced electron transfer (PET) from N,N-dimethylaniline (DMA) to coumarin dyes in a room-temperature ionic liquid (RTIL, [pmim][BF(4)]) and in a mixed micelle containing the RTIL and a triblock copolymer, (PEO)(20)-(PPO)(70)-(PEO)(20), (Pluronic P123) is studied using femtosecond upconversion. A Marcus-like inversion in the rate of PET is observed in neat RTIL. This is attributed to high viscosity and nanostructuring of the RTIL. Diffusion and the rate of PET in the neat RTIL are slower than those in the RTIL-P123 mixed micelle. The coumarin dyes exhibit faster electron transfer and translational diffusion (anisotropy decay) in the RTIL-P123 mixed micelle compared to that in the P123 micelle.

Solvation Dynamics under a Microscope: Single Giant Lipid Vesicle

Picosecond spectroscopy under a confocal microscope is employed to study solvation dynamics of coumarin 153 (C153) inside a single giant lipid vesicle (1,2-dilauroyl-sn-glycero-3-phosphocholine, DLPC) of diameter 20 μm. Fluorescence correlation spectroscopy (FCS) indicates that the diffusion coefficient (D(t)) of the probe (coumarin153, C153) in the immobilized vesicle displays a wide distribution from ~3 to 21 μm(2) s(-1). The distribution of D(t) suggests that the microenvironment of the probe (C153) is highly heterogeneous and the local friction is different for probe molecules in different regions. The values of D(t) is significantly smaller than that for the same dye in bulk water (550 μm(2) s(-1)). This suggests that the probe is located in the interface or membrane region rather than in the water pool of the vesicle. The solvation time of C153 in different regions of the lipid vesicle varies between 750 to 1200 ps. This result clearly shows that a confocal microscope is able to resolve the spatial heterogeneity in local friction (i.e., D(t)) and solvation dynamics within a lipid vesicle.

Binding of Organic Dyes with Human Serum Albumin: A Single‐Molecule Study

Kinetics of binding of dyes at different sites of human serum albumin (HSA) has been studied by single-molecule spectroscopy. The protein was immobilized on a glass surface. To probe different binding sites (hydrophobic and hydrophilic) two dyes, coumarin 153 (C153, neutral) and rhodamine 6G (R6G, cationic) were chosen. For both the dyes, a major (ca. 96-98%) and minor (ca. 3%) binding site were detected. Rate constants of association and dissociation were simultaneously determined from directly measuring fluctuations in fluorescence intensity (τ(off) and τ(on)) and from this the equilibrium (binding) constants were calculated. Fluorescence lifetimes at individual sites were obtained from burst-integrated lifetime analysis. Distributions of lifetime histograms for both the probes (C153 and R6G) exhibit two maxima, which indicates the presence of two binding domains in the protein. Unfolding of the protein has been studied by adding guanidinium hydrochloride (GdnHCl) to the solution. It is observed that addition of GdnHCl affects the dissociation and association kinetics and hence, binding equilibrium of the association of C153. However, the effect of binding of R6G is not affected much. It is proposed that GdnHCl affects the hydrophobic binding sites more than the hydrophilic site.