Increased redox-sensitive green fluorescent protein reduction potential in the endoplasmic reticulum following glutathione-mediated dimerization.

As the endoplasmic reticulum (ER) is the compartment where disulfide bridges in secreted and cell surface proteins are formed, the disturbance of its redox state has profound consequences, yet regulation of ER redox potential remains poorly understood. To monitor the ER redox state in live cells, several fluorescence-based sensors have been developed. However, these sensors have yielded results that are inconsistent with each other and with earlier non-fluorescence-based studies. One particular green fluorescent protein (GFP)-based redox sensor, roGFP1-iL, could detect oxidizing changes in the ER despite having a reduction potential significantly lower than that previously reported for the ER. We have confirmed these observations and determined the mechanisms by which roGFP1-iL detects oxidizing changes. First, glutathione mediates the formation of disulfide-bonded roGFP1-iL dimers with an intermediate excitation fluorescence spectrum resembling a mixture of oxidized and reduced monomers. Second, glutathione facilitates dimerization of roGFP1-iL, which shifted the equilibrium from oxidized monomers to dimers, thereby increasing the molecule's reduction potential compared with that of a dithiol redox buffer. We conclude that the glutathione redox couple in the ER significantly increased the reduction potential of roGFP1-iL in vivo by facilitating its dimerization while preserving its ratiometric nature, which makes it suitable for monitoring oxidizing and reducing changes in the ER with a high degree of reliability in real time. The ability of roGFP1-iL to detect both oxidizing and reducing changes in ER and its dynamic response in glutathione redox buffer between approximately -190 and -130 mV in vitro suggests a range of ER redox potentials consistent with those determined by earlier approaches that did not involve fluorescent sensors.

Hydrogen bonding interpolymer complex formation and study of its host-guest interaction with cyclodextrin and its application as an active delivery vehicle.

Interpolymer complex formation through hydrogen bonding has been investigated between two polymers: poly(acrylamide) (PAAm) and poly(vinyl alcohol) (PVA). The differential properties of the interpolymer complex with varying molecular weights of PVA have been studied by taking three different molecular weights of PVA. Furthermore, the host-guest interaction between the interpolymer complexes prepared and ß-cyclodextrin (ß-CD) has also been studied in detail. PAAm can form interpolymer complexes with PVA because of a cooperative hydrogen bonding interaction. The addition of ß-CD to a dilute aqueous solution of PAAm-PVA results in a competition between interpolymer hydrogen bonding and host-guest interactions. In this article, we have tried to decipher the complex chemistry that occurs in the microheterogeneous solution. The PAAm-PVA binary system and the PAAm-PVA-ß-CD ternary systems have been well characterized by using a fluorescent probe, coumarin-102. Dynamic light scattering (DLS), Fourier transform infrared (FTIR), fluorescence microscopy, and time-resolved fluorescence studies have been performed to substantiate steady-state fluorescence experiments. The results indicate the occurrence of a competitive interaction between the hydrogen bonding of the interpolymer complexes and the host-guest interaction with ß-CD, whereby the later predominates. It is probable that the hydrophobic cavity of ß-CD is threaded with linear polymers, thus forming a macromolecular supraassembly. It has also been concluded that PAAm preferentially interacts with ß-CD by compromising its interaction with PVA. The enhanced deposition and retention of actives with this system was studied with a single species regrowth assay, antibacterial efficacy and the cell viability were studied using the live-dead staining protocol. This therefore opens new avenues in the targeted delivery of actives.

A fully standardized method of synthesis of gold nanoparticles of desired dimension in the range 15 nm-60 nm.

The citrate reduction method of synthesis of gold nanoparticles (AuNPs) as introduced by Frens has been standardized to enable one to prepare AuNPs of desired dimension by controlling the composition of the reactants. The standardization has been made through characterization of the nanoparticles by UV-vis spectroscopy and from the transmission electron microscopic (TEM) measurements. Linearity of the plot of the plasmon absorption maximum (lambda(max)) of the synthesized AuNPs against their diameter as measured from TEM, as well as the plot of lambda(max) with the fractional concentration of citrate in the reaction mixture provides a convenient and easy route to dictate the size of the synthesized AuNPs from a control on the composition of the reactants. The standardization reveals that a calculated composition of citrate (in terms of fractional concentration) in the reaction mixture produces AuNPs of a desired dimension within the range of 15-60 nm. The diameter of the synthesized gold nanoparticles can be confirmed simply from the UV-vis spectrophotometric technique. This essentially makes the use of costly TEM unnecessary, at least for the primary purposes.

Photophysics and dynamics of a ß-carboline analogue in room temperature ionic liquids.

Room temperature ionic liquids are rapidly emerging as a new class of media that are ideally suited for various applications including carrying out chemical reactions. In the present article, we report the photophysics of a ß-carboline analogue, namely, 3-acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine (AODIQ), in three room temperature ionic liquids (RTILs), 1-butyl-3-methylimidazolium methyl sulfate ([BMIM][MeSO(4)]), 1-butyl-3-methylimidazolium octyl sulfate ([BMIM][C(8)SO(4)]) and 1-ethyl-3-methylimidazolium methyl sulfate ([EMIM][MeSO(4)]). Out of these, [BMIM][C(8)SO(4)] is a typical RTIL that forms micellar aggregates above a critical micellar concentration (CMC). Steady state absorption, steady state and time resolved fluorescence techniques are used to probe the properties of these systems. The investigation reveals that the photophysics of AODIQ is modified significantly in the micelle-forming RTIL as compared to that in the other two. A comparative study with the fluorophore in [BMIM][C(8)SO(4)] and a conventional anionic surfactant of a similar hydrophobic chain length from the sodium-n-alkyl sulfate series, viz., sodium octyl sulfate (S(8)S), reveals that the fluorophore experiences a more constrained environment in the RTIL micelle as compared to the conventional anionic micelle.

Electrostatic pushing effect: a prospective strategy for enhanced drug delivery.

Contrary to the expected quenching, unprecedented and remarkable enhancement is observed in the fluorescence of an anionic fluorophore, 8-anilino-1-naphthalene sulfonate, with the addition of bromide ion in cationic cetyltrimethylammonium bromide micellar medium. Electrostatic pushing effect of the halide ion on the anionic fluorophore that forces the probe to penetrate further into the micellar interior has been assigned to be responsible for the novel observation. Experiments with other probes and surfactants propose that the electrostatic pushing effect is rather a general phenomenon. While applying to the ionic drugs in real biosystems, potential application of the unorthodox effect remains in enhancing the solubilization of the drugs into the active target region leading to a radical enhancement in the drug efficacy.

Studies of Triton X-165-beta-cyclodextrin interactions using both extrinsic and intrinsic fluorescence.

The interaction of beta-cyclodextrin with the non-ionic micelle-forming surfactant Triton X-165 (TX-165) has been studied using steady state fluorescence and fluorescence anisotropy techniques. Both extrinsic and intrinsic fluorescence have been exploited for the purpose. Phenosafranin (PSF), a cationic phenazinium dye, has been used as the extrinsic probe while fluorescence of TX-165 has served as the intrinsic one. PSF shows discernible interactions with both TX-165 and beta-CD. The experimental results reveal that the extent of interaction of PSF with TX-165 is greater than with beta-CD. However, addition of beta-CD to a micellar solution of TX-165 containing PSF leads to a disruption of the micelles whereby the fluorophore is released from the micellar environment to the bulk aqueous phase. It has been substantiated that an inclusion complex is formed between the non-ionic surfactant and the cyclodextrin. A 1:1 stoichiometry of the TX-165-beta-CD inclusion complex has been proposed. Such a complexation between TX-165 and beta-CD results in an inhibition in the micellization process of TX-165 leading to an enhancement in the apparent CMC value. The inferences are drawn from a series of experiments, viz., binding studies, determination of micropolarity, heavy-ion quenching studies and steady state fluorescence anisotropy experiments monitoring both extrinsic and intrinsic fluorescences.

Probing the binding interaction of a phenazinium dye with serum transport proteins: a combined fluorometric and circular dichroism study.

In the present investigation, an attempt has been made to study the interaction of phenosafranin (PSF), a cationic phenazinium dye with the transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA), employing steady-state and time-resolved fluorometric and circular dichroism (CD) techniques. The photophysical properties of the dye are altered on binding with the serum proteins. An explicit study with respect to the modification of the fluorescence and fluorescence anisotropy upon binding, effect of denaturant, fluorescence lifetime and CD measurements reveal that the dye binds to both BSA and HSA with almost the same affinity. Far-UV CD spectra indicate a decrease in the percentage of alpha-helicity only for BSA upon binding with the probe. Near-UV CD responses indicate an alteration in the tertiary structure of both the transport proteins because of binding.

Differential interaction of beta-cyclodextrin with lipids of varying surface charges: a spectral deciphering using a cationic phenazinium dye.

Interaction of phenosafranin (PSF), a biologically potent cationic dye molecule, has been studied with zwitterionic and anionic lipid membranes of dimyristoyl-L-alpha-phosphatidylcholine (DMPC) and dimyristoyl-L-alpha-phosphatidylglycerol (DMPG), respectively. The effect of cyclic oligosaccharide, beta-cyclodextrin (beta-CD), on the stability of these probe-bound lipid bilayers has also been investigated exploiting steady state and time-resolved fluorescence, steady state fluorescence anisotropy, and dynamic light scattering techniques. An interpretation of membrane destabilization upon interaction of cyclodextrin with the lipids was drawn exploiting PSF as an extrinsic fluorescent probe. The fluorophore showed discernible interactions with DMPC and DMPG vesicles. Experimental results reveal that the extent of interaction of PSF with DMPG is greater compared to that with DMPC. Addition of beta-CD into the PSF-bound lipids showed a differential effect for the two lipids of varying surface charge characteristics. In the case of DMPC, addition of beta-CD resulted in a preferential interaction of the probe with CD. However, addition of beta-CD to PSF-bound DMPG resulted in the selective interaction of DMPG with the added CD leading to the release of the probe into the bulk aqueous medium.

Binding of a cationic phenazinium dye in anionic liposomal membrane: a spectacular modification in the photophysics.

Interaction of a cationic phenazinium dye, phenosafranin (PSF), with the anionic liposomal vesicle/bilayer of dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) has been demonstrated using steady state and time resolved fluorescence and fluorescence anisotropy techniques. The charge transfer emission spectrum of PSF shows a dramatic modification in terms of fluorescence yield together with an appreciable hypsochromic shift in the lipid environment. The blue shift indicates a lowering in polarity inside the vesicle as compared to that in bulk water. The fluorescence and fluorescence quenching studies and micropolarity determination reveal that the cationic fluorophore has a profound binding interaction with the anionic DMPG membrane. Anisotropy study indicates the imposition of a motional restriction on the probe inside the bilayer. The electrostatic interaction between the cationic dye and the anionic lipid membrane has been argued to be the reason behind all these observations. The results could be useful in analyzing membrane organization and heterogeneity in natural membranes exploiting PSF or alike compounds as fluorescent probes.

Photophysics and rotational relaxation dynamics of cationic phenazinium dyes in anionic reverse micelles: Effect of methyl substitution.

We present here, a detailed photophysical and rotational relaxation dynamical study of three structurally analogous cationic dyes, namely, phenosafranin (PSF), safranin-T (ST), and safranin-O (SO), carried out in well characterized, monodispersed biomimicking anionic reverse micellar nanocavities composed of sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/heptane with increasing water contents. The dyes belong to the phenazinium family and they differ in terms of methyl substitution on the planar phenazinium skeleton. The objective of the present study is to investigate the modification in the photophysical and dynamical behavior of the dyes with the change in the size of the water pool of the reverse micelle and thereby to explore the role of methyl substitution. Steady state and time resolved emission and anisotropy studies have been exploited for the purpose. The dyes are found to exhibit a marked decrease in the fluorescence anisotropy with increasing water/surfactant mole ratio (w), i.e., the water pool size in the reverse micellar core, implying that overall motional restriction experienced by the molecules are decreased with increasing hydration. Some of the depth dependent fluorescence parameters such as fluorescence maximum, fluorescence anisotropy (r) have been monitored for exploring the microenvironment around the probes in the reverse micelles. Fluorescence studies suggest that at low w values, the probes do not penetrate into the reverse micellar core; rather it binds at the interfacial region. Estimates of the micropolarity at the binding sites of the probe molecule have been determined as a function of w. Finally, dynamic studies reveal that both the lifetime and rotational relaxation time decrease with an increase in w for all the three probes, the extent of the decrease being more for PSF than ST and SO. This indicates a stronger binding of the reverse micelle with ST and SO compared to that with PSF which is rationalized in terms of an increase in the hydrophobicity of the former two dyes because of the methyl substitution on the phenazinium moiety.

Excited-state-proton-transfer-triggered fluorescence resonance energy transfer: from 2-naphthylamine to phenosafranin.

Excited-state proton transfer (ESPT) and fluorescence resonance energy transfer (FRET) have been linearly coupled leading to an efficient pH-sensitive energy transfer from 2-naphthylamine (2NA) to a potentially bioactive cationic phenazinium dye, phenosafranin (PSF). The prototropic product produced exclusively from the photoexcited 2NA in the presence of added alkali serves as the donor for the energy transfer process. The energy transfer process is turned on at pH > or = 12, whereas the process is turned off at a pH lower than that. Within the range of pH 12 to 13, the energy transfer efficiency (E) has been shown to follow a linear relation with the solution pH establishing the governing role of pH of the solution on the energy transfer process. The energy transfer follows a long-range dipole-dipole interaction mechanism. The critical energy transfer distance (R0) and the distance between the acceptor and the donor (r) have been determined for the ESPT-promoted FRET process at an optimum pH of 13. The present study involving the coupled processes is simple but has its implication due to its potential to be exploited for designing a pH-sensitive molecular switch.

Deciphering the perturbation of serum albumins by a ketocyanine dye: a spectroscopic approach.

Steady state and time resolved fluorometric and circular dichroism (CD) techniques have been exploited to explore the binding interaction of a ketocyanine dye, namely, 2-[3-(N-methyl-N-phenylamino)-2-propenylidene] indanone (MPAPI) with transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA). The emission spectrum of buffered solution of the dye is found to be perturbed remarkably upon binding with the proteins. An explicit study with respect to modification of fluorescence and fluorescence anisotropy upon binding, effect of denaturant, fluorescence lifetime and CD measurements reveal that the dye binds with both BSA and HSA; the binding being stronger with the latter. Denaturation and CD studies reveal that stability of the proteins increases upon binding with the dye. The probable binding sites of the dye in the proteinous environments have been assessed from fluorescence resonance energy transfer (FRET) study. The probe is argued to be located in the inter domain cleft region of HSA (near Trp-214). From the similarity in the fluorescence behavior of the dye in BSA and HSA it is inferred that in BSA environment the probe is located near Trp-212 rather than Trp-132.

Photophysics and rotational relaxation dynamics of a beta-carboline based fluorophore in cationic alkyltrimethylammonium bromide micelles.

Photophysics and rotational relaxation dynamics of a beta-carboline analog, 3-acetyl-4-oxo-6,7-dihydro-12H-indolo-[2,3-a] quinolizine (AODIQ) have been investigated in cationic alkyltrimethylammonium bromide (nTAB) micelles using steady-state and time-resolved fluorometric techniques. The study reveals modification of its photophysics by the conjugate effect of polarity and rigidity of the micellar environments with varying alkyl chain lengths of the surfactants. Furthermore, it suggests that the fluorophore resides at the micelle-water interfacial domain. Contrary to the single exponential nature of the fluorescence anisotropy decay of AODIQ in aqueous medium, the decay is found to be biexponential in all the micellar environments studied. The enhancements in the steady-state anisotropy and rotational relaxation time in the micellar media compared to that in pure aqueous solution reflect that the fluorophore resides in a motionally restricted environment introduced by the cationic micelles. The rotational correlation time increases marginally with an increase in the surfactant chain length. The rotational relaxation of AODIQ in the micellar environments has been discussed in the light of the two-step and wobbling in a cone model. The model helps in evaluating different rotational parameters and in ascertaining the location of the fluorophore in the micellar media. This technique provides valuable information regarding the rotational relaxations of the fluorophore within an organized assembly. When the lifetime measurements and orientational relaxation measurements are combined, significant inferences can be made regarding the partitioning of the probe in different regions of the micelles.

Photophysics and rotational dynamics of a beta-carboline analogue in nonionic micelles: effect of variation of length of the headgroup and the tail of the surfactant.

Effect of variation of length of nonionic surfactants in terms of the headgroup as well as the tail part on the photophysical and rotational dynamical properties of a beta-carboline analogue, 3-acetyl-4-oxo-6,7-dihydro-12H-indolo-[2,3-a]quinolizine (AODIQ) has been investigated. Steady-state and time-resolved fluorescence and fluorescence anisotropy have been exploited for the purpose. The experiments revealed modification of the photophysics of AODIQ by the conjugate effect of polarity and rigidity of the micellar environments with varying poly(ethylene oxide) chain length in the case of Triton X series and the alkyl chain length in the case of Tween series surfactants. Fluorometric studies suggest that the fluorophore resides at the micelle-water interface in all these systems. The enhancements in the steady-state anisotropy in all the micellar media compared to those in pure aqueous solution reflect that the fluorophore is located in motionally restricted regions introduced by the nonionic micelles. Contrary to the single exponential nature of the fluorescence anisotropy decay of AODIQ in aqueous medium, they were found to be biexponential in the micellar environments. The rotational relaxation of AODIQ in the micellar environments has been discussed in light of the two-step and wobbling in a cone model. The model helps to evaluate different rotational parameters and to ascertain the location of the fluorophore in the micellar media. The significant feature is that the motional restriction decreases with an increase in the poly(ethylene oxide) chain length while it increases with an increase in the alkyl chain length. The difference in the extent of water penetration due to variation in the thickness of the palisade layer and therefore a variation in the micellar polarity with a variation of the length of poly(ethylene oxide) and alkyl chain has been argued to be responsible.

Spectroscopic characterization of phenazinium dye aggregates in water and acetonitrile media: effect of methyl substitution on the aggregation phenomenon.

Absorption, fluorescence, and fluorescence excitation spectral studies of two planar, cationic phenazinium dyes, namely, phenosafranin (PSF) and safranin-T (ST), have been performed in protic and aprotic polar solvents. The studies reveal the formation of both J- and H-aggregates in concentrated solutions. The planarity of the phenazinium skeleton and the presence of a positive charge are attributed to be the driving force for this aggregation behavior. The aggregates are established to be dimers only. The positive inductive effect of the methyl substituents in safranin-T augments the aggregation process. The experiments reveal that for both dyes, the polar protic solvent favors the aggregation process more than the aprotic solvent.

Binding interaction of cationic phenazinium dyes with calf thymus DNA: a comparative study.

Absorption, steady-state fluorescence, steady-state fluorescence anisotropy, and intrinsic and induced circular dichroism (CD) have been exploited to explore the binding of calf thymus DNA (ctDNA) with three cationic phenazinium dyes, viz., phenosafranin (PSF), safranin-T (ST), and safranin-O (SO). The absorption and fluorescence spectra of all the three dyes reflect significant modifications upon interaction with the DNA. A comparative study of the dyes with respect to modification of fluorescence and fluorescence anisotropy upon binding, effect of urea, iodide-induced fluorescence quenching, and CD measurements reveal that the dyes bind to the ctDNA principally in an intercalative fashion. The effect of ionic strength indicates that electrostatic attraction between the cationic dyes and ctDNA is also an important component of the dye-DNA interaction. Intrinsic and induced CD studies help to assess the structural effects of dyes binding to DNA and confirm the intercalative mode of binding as suggested by fluorescence and other studies. Finally it is proposed that dyes with bulkier substitutions are intercalated into the DNA to a lesser extent.

Photophysics of a beta-carboline based non-ionic probe in anionic and zwitterionic liposome membranes.

Interaction of a biologically active beta-carboline based non-ionic probe, 3-acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine (AODIQ), with the liposomal vesicles of dimyristoyl-l-alpha-phosphatidylcholine (DMPC) and dimyristoyl-l-alpha-phosphatidylglycerol (DMPG) has been demonstrated using steady-state and time-resolved fluorescence and fluorescence anisotropy techniques. Polarity sensitive intramolecular charge transfer of AODIQ shows a large hypsochromic shift along with an enhancement in the fluorescence quantum yield and fluorescence lifetime in the bilayer membranes compared to those in aqueous buffer solution. Polarity of the immediate vicinity of the probe in the lipid environments has been determined. The fluorometric, quenching and micropolarity determination studies reveal that the fluorophore penetrates deeper in the zwitterionic DMPC membrane compared to the anionic DMPG vesicle. Enhancement in the rotational relaxation time of AODIQ in liposomal membranes suggests that the fluorophore exists in motionally restricted environments.

Application of anionic micelle for dramatic enhancement in the quenching-based metal ion fluorosensing.

We introduce a simple and efficient strategy to enhance the efficiency of a quenching-based fluorosensor for metal ions by several orders of magnitude by using commercially available anionic surfactants varying hydrophobic chain length. Anionic surfactants with a proper choice of hydrophobic chain length at their optimum concentrations are efficient to boost up the efficiency of copper ion sensor dramatically. This simple and convenient strategy is, in general, applicable to quenching-based fluorosensors, new or established, in aqueous solution. It is powerful enough to transform a virtually non-sensor fluorophore to a sensor with a commendable efficiency with the help of proper surfactant. Thus, in this communication, light has been thrown on the application of surfactants toward increasing fluorosensing efficiency of a quenching based sensor.