STUDY OF ORGANIZED MEDIA USING FEMTOSECOND SPECTROSCOPY AND CONFOCAL MICROSCOPY

Self-organized nanostructures play a crucial role in a wide range of natural phenomena ranging from molecular recognition (e.g. enzyme-substrate, antigenantibody interaction), to a more general case of unusual chemistry in confined environments. Due to the confinement and local interaction, the biological activity and dynamics in these assemblies are significantly different from those in ordinary solutions. In recent years, ultrafast time resolved spectroscopy revealed substantial amount of new information on these systems.The experimented results has stimulated a large body theoretical studies and in particular, large scale computer simulations. The main motivation of this thesis is to understand the role of confinement on the spectroscopy and dynamics with both temporal and spatial resolution. In the present thesis, we studied different organized media using femtosecond upconversion, time resolved confocal microscopy and fluorescence correlation spectroscopy (FCS). In bulk, all chemical informations are averaged over an exceedingly large number of molecules of the order of the Avogadro number. Recent single molecule studies have demonstrated variation of properties of individual molecular systems in an ensemble. This has completely revolutionized our understanding of chemistry.The research was carried out under the supervision of Prof. Kankan Bhattacharyya of the Physical Chemistry division under SCS [School of Chemical Sciences]The research was conducted under CSIR fellowship and DST research gran

Role of a mild reducing agent in designing a copper nanocluster-based tunable dual-metal sensor for the selective and sensitive detection of Ag+ and Fe2+

Protein-protected metal nanoclusters emerged as a promising class of biofriendly nano-materials due to their interdisciplinary applications, namely as efficient metal sensors. Here we have successfully designed a photoluminescent protein (lysozyme) scaffolded copper nanocluster (Lys-Cu NC) (λex= 365 nm, λem= 437 nm) that can be used as a cost-effective dual metal sensor for the sensitive and selective detection of silver (Ag+) and ferrous (Fe2+) ions through two independent photoluminescence turn off mechanisms. The nanocluster when synthesized in the presence of a mild reducing agent, hydrazine (N2H4), can selectively detect Ag+ (LOD = 4 nM) through a size-induced photoluminescence quenching method involving both static and dynamic mechanisms. By removing the excess N2H4 from the medium, the selectivity can be tuned towards Fe2+, which can quench the photoluminescence intensity through a static charge transfer process. However, the selectivity towards Ag+ can be recovered by externally adding N2H4 again to the medium. Thus, the selectivity of the nanocluster can be switched back and forth between Ag+ and Fe2+ by simply controlling the redox properties of the medium. This simple method of designing a dual-metal sensor reported in this study is unique and can be further extended to design multi-metal sensors for specific biologically and environmentally relevant metal ions in the future

A fluorescence correlation spectroscopy study of the diffusion of an organic dye in the gel phase and fluid phase of a single lipid vesicle

The mobility of the organic dye DCM (4-dicyanomethylene-2-methyl-6-p-dimethyl aminostyryl-4H-pyran) in the gel and fluid phases of a lipid vesicle is studied by fluorescence correlation spectroscopy (FCS). Using FCS, translational diffusion of DCM is determined in the gel phase and fluid phase of a single lipid vesicle adhered to a glass surface. The size of a lipid vesicle (average diameter 100 nm) is smaller than the diffraction limited spot size (250 nm) of the microscope. Thus, the vesicle is confined within the laser focus. Three lipid vesicles (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)) having different gel transition temperatures (-1, 23, and 41°C, respectively) were studied. The diffusion coefficient of the dye DCM in bulk water is 300 μ m2/s. In the lipid vesicle, the average Dt decreases markedly to ~5 μm2/s (60 times) in the gel phase (for DPPC at 20°C) and 40 μm2/s (~8 times) in the fluid phase (for DLPC at 20°C). This clearly demonstrates higher mobility in the fluid phase compared with the gel phase of a lipid. It is observed that the Dt values vary from lipid to lipid and there is a distribution of Dt values. The diffusion of the hydrophobic dye DCM (Dt~ 5 μ m2/s) in the DPPC vesicle is found to be 8 times smaller than that of a hydrophilic anioinic dye C343 (Dt~ 40 μ m2/s). This is attributed to different locations of the hydrophobic (DCM) and hydrophilic (C343) dyes

Rapid Detection of Ag (I) via Size Induced Photoluminescence Quenching of Biocompatible Green Emitting L-Tryptophan Scaffolded Copper Nanocluster

Atomically precise metal nanoclusters capped with small molecules like amino acids are highly favoured due to their specific interactions and easy incorporation into biological systems. However, they are rarely explored due to the challenge in surface functionalization of nanocluster with small molecules. Herein, we report the synthesis of green emitting (λ_ex = 380 nm, λ_em = 500 nm) single amino acid (L-tryptophan) scaffolded copper nanocluster (Trp-Cu NC) via one-pot route under mild reaction conditions. The synthesized nanocluster can be used for the rapid detection of heavy metal (Ag(I)) in the nanomolar concentration range in real environmental and biological samples. The strong green photoluminescence intensity of the nanocluster quenched significantly upon addition of Ag(I) due to the formation of bigger nanoparticles, thereby losing its energy quantization. A notable colour change from light yellow to reddish brown can also be observed in the presence of Ag(I) allowing its visual colorimetric detection. Portable paper strips fabricated with Cu-Trp NC can be reliably used for on-site visual detection of Ag(I) in the micromolar concentration range. The Trp-Cu NC possesses excellent biocompatibility making them suitable nanoprobe for cell imaging, thus can act as an in-vivo biomarker. The nanocluster showed a significant spectral overlap with an anticancer drug doxorubicin, thus can be used as an effective FRET pair. FRET results can reveal important information regarding the attachment of the drug to the nanocluster and hence, its role as a potential drug carrier for targeted drug delivery within human body

Lysozyme protected Cu Nano-Cluster: A Photo Switch for the Selective Sensing of Iron (Fe2+)

Protein capped metal nanoclusters gained a lot of recent attention due to their wide range of applications. However copper nanoclusters are difficult to synthesize due to their tendency to undergo oxidation. Here we successfully synthesized a stable, biocompatible lysozyme protected Cu nanocluster (Lys-Cu NC) using an optimized green one-pot protocol under aqueous condition at room temperature. The nanocluster showed a strong photoluminescence intensity (λex = 365 nm, λem = 430 nm) which can be significantly and selectively quenched (off) by Fe2+ ions. Upon addition of NaOH the initial photoluminescence intensity can be recovered completely (on) thereby making the nanocluster a suitable candidate for a photo switch that can be reliably reused for the selective and sensitive detection of Fe2+ ions in the nanomolar (detection limit ~2.5 nM) concentration range. The synthesized nanocluster further has been successfully used to rapidly estimate iron level in complex systems (such as ground water and human hemoglobin samples). The photoluminescence intensity of the nanocluster is also sensitive towards temperature indicating that it can be used as a temperature sensor in different biological systems. This biofriendly nanocluster further used as an excellent nanoprobe for in-vivo cell imaging studies. Thus this Lys-Cu NC can be emerged as a next generation novel nanoprobe for various interdisciplinary applications

Deuterium isotope effect on femtosecond solvation dynamics in an ionic liquid microemulsion: an excitation wavelength dependence study

The deuterium isotope effect on the solvation dynamics and the anisotropy decay of coumarin 480 (C480) in a room temperature ionic liquid (RTIL) microemulsion is studied by femtosecond up-conversion. The microemulsion consists of the RTIL 1-pentyl-3-methyl-imidazolium tetra-fluoroborate ([pmim][BF4]) in triton X-100 (TX-100)/benzene. Replacement of H2O by D2O in the microemulsion causes retardation of solvation dynamics. The average solvation time of C480 (τs) in RTIL microemulsion with 5 wt % D2O is 1.5-1.7 times slower compared to that in the H2O containing RTIL microemulsion. This suggests that the main species in the microemulsion responsible for solvation is the water molecules. In both D2O and H2O containing RTIL microemulsion, the solvation dynamics exhibits marked dependence on the excitation wavelength (λ ex) and becomes about 15 times faster as λ ex increases from 375 to 435 nm. This is ascribed to the structural heterogeneity in the RTIL microemulsion. For λ ex = 375 nm, the region near the TX-100 surfactant is probed where bound water molecules cause slow solvation dynamics. At 435 nm, the RTIL pool is selected where the water molecules are more mobile and hence gives rise to faster solvation. The average time constant of anisotropy decay shows opposite dependence on λ ex and increases about 2.5-fold from 180 ps at λ ex = 375 nm to 500 ps at λ ex = 435 nm for D2O containing RTIL microemulsion. The slower anisotropy decay at λ ex = 435 nm is ascribed to the higher viscosity of RTIL which causes greater friction at the core

Effect of ionic liquid on diffusion in P123 gel: fluorescence correlation spectroscopy

The effect of two room-temperature ionic liquids (RTILs) on the diffusion of three fluorescent dyes in the gel phase of a triblock copolymer, (PEO)<SUB>20</SUB>-(PPO)<SUB>70</SUB>-(PEO)<SUB>20</SUB> [Pluronic P123; poly ethylene oxide (PEO), poly propylene oxide (PPO)], was studied by using fluorescence correlation spectroscopy (FCS). We used three dyes, 4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran (DCM), coumarin 480 (C480), and coumarin 343 (C343). By field-emission scanning electron microscopy (FESEM), it was observed that the macroscopic structure of the P123 gel remained unaffected upon addition of RTIL. In the absence of RTIL, the diffusion coefficient (D<SUB>t</SUB>) of the hydrophobic dye DCM (1 μm<SUP>2</SUP>s<SUP>-1</SUP> at the core) is smaller than that of the other two hydrophilic dyes (7μm<SUP>2</SUP>s<SUP>-1</SUP> for C480 and C343). On addition of RTIL, the D<SUB>t</SUB> values of all of the dyes increase, indicating a decrease in local viscosity (η<SUB>eff</SUB>). The η<SUB>eff</SUB> of the core of the RTIL-P123 gel estimated from the D<SUB>t</SUB> of DCM is lower than that of both the P123 gel (at the core η =90 cP) and RTIL (η =110 cP). It is shown that the RTIL affects the structure of the gel by modifying the size of the micellar aggregates and by penetrating the core

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] [BF4]) 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] [BF4]). 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 H2O by D2O causes an increase in the time constant of the ultraslow rise to 2350 ps

Diffusion of organic dyes in immobilized and free catanionic vesicles

Fluorescence correlation spectroscopy (FCS) has been used to study the motion of fluorescent dyes in a giant (diameter 20000 nm = 20 μm) catanionic vesicle comprised of the surfactant sodium dodecyl sulfate (SDS) and dodecyltrimethyl ammonium bromide (DTAB). The diffusion in the anion (SDS) rich catanionic vesicle was studied both in bulk water and in an immobilized vesicle attached to a positively charged glass surface. In the case of the immobilized vesicle, the diffusion coefficients (Dt) of R6G (rhodamine 6G), DCM (4-dicyanomethylene-2-methyl-6-p-dimethyl aminostyryl-4H-pyran), and C343 (coumarin 343) are found to be 1.5, 2.5, and 10 μ m2/s, respectively, which are 280, 120, and 55 times slower compared to those for the same dyes in bulk water. The magnitude of Dt is found to vary for different vesicles. This was attributed to the difference in size and shape of the immobilized vesicles. In bulk, R6G binds completely to the vesicle and exhibits extremely slow diffusion with Dt=0.5 ± 0.1μm2/s (850 and 3 times slower compared to that of R6G in bulk water and within the immobilized vesicle). This is attributed to very slow overall diffusion of the very large size vesicles (20 μ m = 20000 nm). Both of the dye molecules (DCM and C343) show two different diffusion coefficients for the vesicles in bulk. In this case, the small Dt (0.5±0.1 μm2/s) corresponds to the diffusion of the vesicle as a whole and the large Dt value (300 and 550 μm2/s for DCM and C343, respectively) corresponds to the free dye molecules in bulk water