High-throughput screening of enzyme libraries: thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments

Single bacterial cells, each expressing a different library variant, were compartmentalized in aqueous droplets of water-in-oil (w/o) emulsions, thus maintaining a linkage between a plasmid-borne gene, the encoded enzyme variant, and the fluorescent product this enzyme may generate. Conversion into a double, water-in-oil-in-water (w/o/w) emulsion enabled the sorting of these compartments by FACS, as well as the isolation of living bacteria cells and their enzyme-coding genes. We demonstrate the directed evolution of new enzyme variants by screening >10(7) serum paraoxonase (PON1) mutants, to yield 100-fold improvements in thiolactonase activity. In vitro compartmentalization (IVC) of single cells, each carrying >10(4) enzyme molecules, in a volume of <10 femtoliter (fl), enabled detection and selection despite the fast, spontaneous hydrolysis of the substrate, the very low initial thiolactonase activity of PON1, and the use of difusable fluorescent products.     

Chemically Modified Human Immunoglobulin G: Hydrophobicity and Surface Activity at Air/Solution Interface

Covalent modification of human IgG by fatty acid esters (C8 and C16) of N-hydroxysuccinimide was carried out. Surface hydrophobicity measurements, using the fluorescent probe 8-anilino-1-naphthalenesulfonate, indicate an increase in the surface protein hydrophobicity with an increase in the number and in the length of the attached alkyl chains. The modified IgGs decrease surface tension at the air/solution interface more effectively than the native protein. The values of the molecular cross-sectional areas (DeltaA) estimated from the kinetic data are in the range of 100-300 ?2 and reflect the size of protein segments at the interface during the adsorption process. About 40-50% increase in the DeltaA was observed upon attachment of the C8 groups to the native IgG. The lengthening of the bound alkyl chain from C8 to C16 results in a further increase in this value. The influence of the overall IgG hydrophobicity and the length of the attached alkyl chain on the dimensions of the mobile protein segment at the surface are discussed. Copyright 1999 Academic Press.     

Structure of microemulsions with gemini surfactant studied by solvatochromic probe and diffusion NMR

The structure of microemulsions prepared by the anionic gemini surfactant didodecyl diphenyl ether disulfonate (C12-DADS) was investigated by a solvatochromic probe and nuclear magnetic resonance (NMR) diffusion measurements. The NMR measurements indicate the presence of bicontinuous and oil-in-water microemulsions depending on microemulsion composition. The absorbance spectra of the solvatochromic probe, Nile red, indicate the solubilization of the probe in different sites, in agreement with the NMR findings. It was also found that the microemulsions were capable of dissolving the hydrophobic probe, Nile red, up to four times better than expected if it were simply dissolved in the toluene phase.     

Synthesis of copper nanoparticles catalyzed by pre-formed silver nanoparticles

Synthesis of well dispersed copper nanoparticles was achieved by reduction of copper nitrate in aqueous solution using hydrazine monohydrate as a reducer in the presence of preformed silver nanoparticles as catalysts. It has been demonstrated that addition of silver nanoparticles to the reaction mixture leads to formation of aqueous dispersion of copper nanoparticles and also results in a drastic reduction in reaction time compared to procedures reported in the literature. The absorption spectrum of the dispersions, HR-TEM and STEM images and XRD pattern indicate the formation of copper nanoparticles with particle size in the range of 5–50 nm.     

Ring stain effect at room temperature in silver nanoparticles yields high electrical conductivity

We demonstrate that metallic rings formed spontaneously at room temperature via evaporation of aqueous drops containing silver nanoparticles (20-30 nm in diameter) exhibit high electrical conductivity (up to 15% of that for bulk silver). The mechanism underlying this self-assembly phenomena is the "ring stain effect", where self-pinning is combined with capillary flow to form a ring consisting of close-packed metallic nanoparticles along the perimeter of a drying droplet. Our macroscopic and microscopic (applying conductive atomic force microscopy) transport measurements show that the conductivity of the ring, which has a metallic brightness, is orders of magnitude larger than that of corresponding aggregates developed without the ring formation, where high conductivity is known to appear only after annealing at high temperature.     

Flow cytometry: a new method to investigate the properties of water-in-oil-in-water emulsions

We present a new and facile method to evaluate w/o/w emulsions containing fluorescent markers by flow cytometry. Flow cytometry allows simultaneous measurement of w/o/w emulsion droplets "marked" with a fluorescent marker or "blank" without the need for complicated sample preparation. The yield of preparation of the w/o/w emulsion and the release rate of the fluorescent marker FITC-BSA were investigated by this new method. The release fraction (after 24 h) of FITC-BSA from the w/o/w emulsion decreased with increasing concentration of FITC-BSA inside the internal phase, just like the release fraction of NaCl as marker from the w/o/w emulsion. Flow cytometry results show that the yield and release behavior in w/o/w emulsions are in agreement with results reported by more complicated methods.     

FORMATION OF OIL IN GLYCEROIVWATER EMULSIONS: EFFECT OF SURFACTANT ETHYLENE OXIDE CONTENT

Oil-in-glycerol/water emulsions at various ratios of water to glycerol in the external phase were prepared with polyoxyethylated octylphenols and light mineral oil. As the water concentration in the external phase decreased, oil droplet size decreased down to a minimum size beyond which oil separation occurred. Also, the cloud points of various surfactants were depressed toward room temperature as the water content of the glycerol/water mixtures decreased. It was possible therefore to correlate the concentration of water needed for formation of the smallest droplets to the concentration of water needed for depression of the cloud point of each surfactant to room temperature.     

Photostabilization of an entomopathogenic fungus using composite clay matrices

To provide photostabilization for entomopathogenic fungi by anionic dyes, composite matrices based on clay-biopolymer combinations were prepared. In the first step, the negative surface charge of various clays (montmorillonite, attapulgite, bentonite and kaolinite) was reversed to positive by adsorption to the polycationic biopolymer chitosan. The second step involved adsorption of the toxicologically safe anionic dyes fast green (FG) and naphthol yellow S (NYS) to the clay complexes. Compared with cytotoxic photoprotectants like berberine, palmatine and acriflavine, the anionic dyes have no adverse effects up to a concentration of 1 M. In assays using various clay-chitosan-dye matrices and UV irradiation from a lamp source, it was evident that both FG and NYS provided considerable photostabilization for conidia of the entomopathogenic fungus Aschersonia spp. that served as a model biocontrol agent. Apparently, because of the light-dispersing property, bentonite and attapulgite per se provided significant photoprotection. All clay matrices containing FG provided a substantial photostabilization effect.     

Ultrasound-assisted dispersion of SrFe<Subscript>12</Subscript>O<Subscript>19</Subscript> nanoparticles in organic solvents and the use of the dispersion as magnetic cosmetics

A new method of dispersing the aggregated strontium hexaferrite (SrFe₁₂O₁₉) magnetic nanoparticles in organic solvents such as propylene glycol monomethyl ether acetate (PGMEA), propylene glycol (PG), and glycerol, by an ultrasonic bath is reported herein. The particles size of SrFe₁₂O₁₉ after treatment with the PGMEA is in the range 70–100 nm. The structure of dispersed SrFe₁₂O₁₉ was characterized using transmission electron microscopy (TEM), high resolution scanning electron microscopy (HR SEM) and thermo gravimetric analysis (TGA). This dispersed material was used for the preparation of a topical magnetic cosmetic product as follows: The dispersion of SrFe₁₂O₁₉ in PG was mixed with “Dermud-Ahava Body Cream”, an ‘oil in water’ emulsion of a Dead Sea mineral cosmetic, “AHAVA”, and the magnetic properties of the created composite were determined. The ferrimagnetic behavior of the composite has been demonstrated as being very similar to the behavior of strontium hexaferrite itself.