Anionic Fluorophore Research Articles

Fluorescence Characterization of DDAB−AOT Catanionic Vesicles

We have used fluorescence and light scattering measurements to characterize the unilamellar vesicles formed in aqueous solution from binary mixtures of the cationic surfactant didodecyldimethylammonium bromide (DDAB) and the anionic surfactant sodium dioctylsulfosuccinate (Aerosol OT or AOT). We focus our study on the early phase of catanionic vesicle formation after surfactant mixing. With two complementary fluorescence probes, the anionic fluorophore TNS (2-(p-toluidino)naphthalene-6-sulfonate) and the neutral probe prodan (6-propionyl-2-(dimethylamino)-naphthalene), we observe the creation of a mixed vesicle via the rapid incorporation of the anionic surfactant AOT (present as surfactant monomers in solution) into DDAB vesicles. Deconvolutions of the overall probe fluorescence emission spectra differentiate among the net surface charges on DDAB−AOT vesicles of varying composition. Furthermore, when TNS is added to DDAB-rich mixed vesicles, we observe time-dependent fluorescence variations that are cons...

Nitrosative stress induced cytotoxicity in Giardia intestinalis.

To investigate the antigiardial properties of the nitrosating agents: sodium nitrite, sodium nitroprusside and Roussin's black salt. Use of confocal laser scanning microscopy and flow cytometry indicated permeabilization of the plasma membrane to the anionic fluorophore, DiBAC4(3) [bis(1,3-dibutylbarbituric acid) trimethine oxonol]. Loss of plasma membrane electrochemical potential was accompanied by loss of regulated cellular volume control. Changes in ultrastructure revealed by electron microscopy and capacity for oxygen consumption, were also consequences of nitrosative stress. Roussin's black salt (RBS), active at micromolar concentrations was the most potent of the three agents tested. These multitargeted cytotoxic agents affected plasma membrane functions, inhibited cellular functions in Giardia intestinalis and led to loss of viability. Nitrosative damage, as an antigiardial strategy, may have implications for development of chemotherapy along with suggesting natural host defence mechanisms.

Light-Harvesting Supramolecular Hydrogels Assembled from Short-Legged Cationic L-Glutamate Derivatives and Anionic Fluorophores

Supramolecular hydrogels (see Figure for structure) are spontaneously formed by mixing aqueous dispersions of short-legged ammonium amphiphiles and fluorescent anions. Energy transfer between donor (naphthalene) and acceptor (anthracene) anions is shown to be more efficient in the hydrogels compared to that in dilute aqueous bilayer dispersions.

Glycine blocks opening of a death channel in cultured hepatic sinusoidal endothelial cells during chemical hypoxia.

Using confocal microscopy, we investigated mechanisms underlying loss of plasma membrane integrity during necrotic death of cultured hepatic sinusoidal endothelial cells exposed to 2.5 mM potassium cyanide (chemical hypoxia). After 2-3 h, the anionic fluorophore calcein abruptly began to enter the cytosol, and nuclei labeled with cationic propidium after another 2-5 min. As calcein permeated, growth of blebs on the plasma membrane accelerated. Lucifer yellow, another anionic fluorophore, entered identically to calcein, whereas high molecular weight dextrans (40-2000 kDa) entered like propidium. Glycine slowed, but did not prevent calcein entry, whereas permeation of propidium and high molecular weight dextrans was blocked completely by glycine. These findings suggest that opening of a glycine-sensitive organic anion channel, or death channel, precipitates a metastable state characterized by rapid cell swelling and bleb growth. This metastable state culminates in non-specific breakdown of the plasma membrane permeability barrier and irreversible cell death.

PH-Insensitive Ion Selective Optode: A Coextraction-Based Sensor for Potassium Ions

A novel type of optical potassium sensor is presented whose response mechanism is virtually insensitive to pH and ionic strength. It is based on a coextraction mechanism using a lipophilic ion carrier and a fluorescent anion, the two contained in a solid oil-in-water emulsion. The latter is composed of lipophilic plasticizer droplets (containing valinomycin) and a hydrogel containing the highly solvatochromic anionic dye merocyanine 540. On exposure to a solution of potassium ions, these are extracted, along with the anionic fluorophore, into the plasticizer phase wherein the dye is much more fluorescent than in hydrogel. The dynamic range of sensor response can be adjusted by variation of the membrane composition. Membranes were optimized for measurements in the physiological range. The dynamic range is from 0.1 to 50 mmol/L K+. Response times are <3 min within this concentration range.

Multilayer Potassium Sensor Based on Solid-State Coextraction

A highly selective solid state multilayer sensor is presented which enables fluorescence optical determination of potassium ions. It is based on co-extraction using valinomycin along with a fluorescent anion, both contained in different layers. The sensor membrane is composed of two layers of highly different lipophilicity. The lipophilic phase contains the carrier valinomycin whilst the hydrophilic phase contains the anionic fluorophore sulforhodamin B. On exposure to potassium ions, the lipophilic phase becomes fluorescent after coextraction of both potassium ions and the anionic fluorophore. To prevent leaching of the dye, an additional blocking layer (nafion) was spread on the sensor. The dynamic range is from 0.1 to 50 mmol/l K + with very low cross-sensitivity to pH and ionic strength. The response times vary from 1 to 2 h.

Spatially Reversible Electric Field-Induced Permeation in Pure and Mixed Langmuir-Blodgett Layers of Hemicyanine Dyes and Octadecanoic Acid on Poly(2-Vinylpyridine)

AbstractLangmuir-Blodgett (LB) films of the hemicyanine dyes 4-(4-dihexadecylaminostyryl)- N-methylpyridinium iodide and 4-(4-dioctylaminostyryl)-N-propylsulfonate pyridinium mixed with octadecanoic acid have been deposited onto H2O-swollen poly(2-vinylpyridine) (P2VP) as mass transport barriers. The transport properties of these structures have been studied both with and without the application of an electric field in the range 0 ≤ E ≤ 100 V/cm, by observing the rate of permeation of both anionic (fluorescein) and zwitterionic (rhodamine B) fluorophores. The apparent diffusion coefficient at E = 0 decreases exponentially with the number of monolayers through which the probe molecule must permeate, in agreement with a hole-mediated permeation model. Application of an external electric field is observed to control the rate and direction of the mass transport of the probe molecule across the barrier layer. The electric field effect is both spatially reversible, i.e. the probe can be transported in either direction across the barrier layer, and temporally reversible, i.e. the permeability cycles with the field between large values when the field is on and small values when it is not applied. The results are consistent with an interpretation in which electroporation is the dominant mechanism for transport in the field-on state.

Biological membrane modeling with a liquid/liquid interface. Probing mobility and environment with total internal reflection excited fluorescence

Total internal reflection of exciting light, in combination with fluorescence intensity and polarization measurements, was used to selectively study fluorescent compounds adsorbed to the interface region between two immiscible liquids. A fluorometer was constructed which provided excitation at variable angles of incidence and allowed sensitive detection of polarized fluorescence emitted from the interface. The compound 4,4'-bis-1-phenylamino-8-naphthalenesulfonate (bis-ANS) was examined at a decalin/water interface and was found to possess remarkable affinity for the interface region with the bulk of the adsorbed molecule residing in the decalin phase. The adsorbed fluorophore displayed an apparent hindered rotation in the plane of the interface with a rotational diffusion coefficient 3- to 12-fold lower than that expected for bis-ANS in solution. While other dyes examined were not found to be significantly surface active, the addition of cationic surfactant sufficed to induce adsorption of the anionic fluorophore 1-aminonaphthalene-3,6,8-trisulfonic acid. This fluoropore was found to reside in an aqueous environment when bound to the interface, and it also exhibited hindered rotation in the plane of the interface. As the concentrations of the dyes were increased, both adsorbed dyes exhibited polarization reductions consistent with excitation energy transfer. Adsorption of bis-ANS was reversed by addition of bovine serum albumin. The membrane protein cytochrome b5 was found not to bind at the decalin/water interface, indicating that interaction with lipid is required for its adherence to biological membranes.

Weak acid-induced release of liposome-encapsulated carboxyfluorescein

Leakage of the entrapped anionic fluorophore carboxyfluorescein was used as a measure of the permeability of liposomes to several different acids. Carboxyfluorescein leakage increased with increasing buffer concentration at a given pH and depended on its chemical nature: apolar weak acids such as acetic or pyruvic acids induced fast leakage at relatively high pH (4 to 5), while glycine, aspartic, citric and hydrochloric acids induced leakage only at lower pH. Fluorescence leakage measurements reflected the acidification of the liposomes' aqueous spaces, which was primarily caused by the diffusion of undissociated acid molecules across the lipid bilayer. A simple mathematical model in accord with this hypothesis and assuming that carboxyfluorescein leakage was directly related to the proportion of its neutral lactone form, described satisfactorily the carboxyfluorescein leakage kinetics and allowed rough estimation of permeability coefficients for carboxyfluorescein (neutral lactone form; 9 · 10 −9 cm · s −1), acetic acid ( >1 · 10 −7 cm · s −1 ) and glycine (cation: 6 · 10 −9 cm · s −1). These results are consistent with low effective proton permeability of liposomes ( <5 · 10 −12 cm · s −1 ) and with the permeability coefficient of HCl (3 · 10 −3 cm · s −1) reported by Nozaki and Tanford ((1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4324–4328). Diffusion of weak acid molecules across lipid membranes has implications for drug encapsulation and delivery, and may be of biological significance.