Individual Coating of Entomopathogenic Nematodes with Titania (TiO2) Nanoparticles Based on Oil-in-Water Pickering Emulsion: A New Formulation for Biopesticides.

This study presents a new eco-friendly formulation of entomopathogenic nematodes (EPNs) based on individual coating of EPNs with titanium dioxide (TiO2) nanoparticles (NPs) and mineral oil via oil-in-water Pickering emulsions. Mineral oil-in-water emulsions stabilized by amine-functionalized titanium dioxide (TiO2-NH2) particles were prepared. 40:60 and 50:50 oil-water volume ratios using 2 wt % TiO2-NH2 particles were found to be the most stable emulsions with a droplet size suitable for the formulation and were further studied for their toxicity against the incorporated EPNs. Carboxyfluorescein was covalently bonded to TiO2-NH2 NPs, and the resulting composite was observed via fluorescence confocal microscopy. The dry coating was evaluated using SEM and confocal microscopy, which showed significant nematode coverage by the particles and oil. The final formulation was biocompatible with the studied EPNs, where the viability of the EPNs in the formulation was equivalent to control aqueous suspension after 120 days. Finally, yields of nematodes from infected Galleria mellonella cadavers collected for 150 days showed no significant differences (P > 0.05) using the tested emulsions compared to the control containing nematodes in water.

Platform for Active Vaccine Formulation Using a Two-Mode Enhancement Mechanism of Epitope Presentation by Pickering Emulsion.

The efficiency of epitope-based vaccination (subunit vaccines) is tightly correlated with heterogeneity and the high density of epitope presentation, which maximizes the potential antigenic determinants. Here, we developed a two-mode platform for intensifying the epitope presentation of subunit vaccines. The two-mode epitope presentation enhancement includes a covalent attachment of high concentrations of SARS-CoV-2-S1 peptide epitope to the surface of virus-like-particles (VLPs) and the subsequent assembly of VLP/epitope conjugates on the oil droplet surface at an oil/water interface of an emulsion as Pickering stabilizers. The resultant emulsions were stable for weeks in ambient conditions, and our platform was challenged using the epitope of the SARS-CoV-2-S1 peptide that served as a model epitope in this study. In vivo assays showed that the αSARS-CoV-2-S1 immunoglobulin G (IgG) titers of the studied mouse antisera, developed against the SARS-CoV-2-S1 peptide under different epitope preparation conditions, showed an order of magnitude higher IgG titers in the studied VLP-based emulsions than epitopes dissolved in water and epitopes administered with an adjuvant, thereby confirming the efficacy of the formulation. This VLP-based Pickering emulsion platform is a fully synthetic approach that can be readily applied for vaccine development to a wide range of pathogens.

Encapsulation of Bacillus thuringiensis in an inverse Pickering emulsion for pest control applications.

Here, we present an inverse Pickering emulsion-based formulation for Bacillus thuringiensis serovar aizawai (BtA) encapsulations utilized towards pest control applications. The emulsification was carried out by high shear homogenization process via ULTRA-TURRAX®. The water-in-mineral oil emulsions were stabilized by commercial hydrophobic silica. Different silica contents and water/oil ratios were studied. Stable emulsions were obtained at 2 and 3 wt% silica at 30% and 20% water volumes, respectively. The structure of the Pickering emulsions were characterized by laser scanning confocal microscopy and cryogenic scanning electron microscopy. The BtA cells, spores and crystals were encapsulated in the water droplets of the inverse Pickering emulsions. An emulsion composed of 3 wt% silica and 30% water was found to be the most suitable for encapsulation. The pest control efficiency of the encapsulated BtA against Spodoptera littoralis first instar larvae was tested. The studied BtA/emulsion system exhibited a mortality rate of 92%. However, the non-formulated BtA has shown 71% mortality, and the emulsion alone resulted in only 9% mortality. These findings confirm that an emulsion with encapsulated BtA can function as an efficient formulation for biopesticides.

Single-Conidium Encapsulation in Oil-in-Water Pickering Emulsions at High Encapsulation Yield.

This study presents an individual encapsulation of fungal conidia in an oil-in-water Pickering emulsion at a single-conidium encapsulation yield of 44%. The single-conidium encapsulation yield was characterized by analysis of confocal microscopy micrographs. Mineral oil-in-water emulsions stabilized by amine-functionalized titania dioxide (TiO2-NH2 or titania-NH2) particles were prepared. The structure and the stability of the emulsions were investigated at different compositions by confocal microscopy and a LUMiSizer® respectively. The most stable emulsions with a droplet size suitable for single-conidium encapsulation were further studied for their individual encapsulation capabilities. The yields of individual encapsulation in the emulsions; i.e., the number of conidia that were individually encapsulated out of the total number of conidia, were characterized by confocal microscopy assay. This rapid, easy to use approach to single-conidium encapsulation, which generates a significantly high yield with eco-friendly titania-based emulsions, only requires commonly used emulsification and agitation methods.

Covalent Immobilization of Polyaniline Doped with Ag+ or Cu2+ on Carbon Nanotubes for Ethylene Chemical Sensing.

Multi-walled carbon nanotubes (MWCNTs) are promising materials for chemical gas sensing because of their high electrical and mechanical properties and significant sensitivity to changes in the local environment. However, high-content MWCNT films suffer from the low tunability of the electrical resistance, which is crucial for high chemoresistive sensing performance. This study reports the conjugation of MWCNTs and oligomers of polyaniline (PANI) doped with Ag+ or Cu2+ incorporated into a PVC/polyacrylate. MWCNTs were sonicated in n-methyl pyrrolidine (NMP), and PANI was conjugated via a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and an N-hydroxysuccinimide (EDC/NHS) process. MWCNT/PANI Ag+ or Cu2+ conjugates were doped to form a coordinate bond. The doped conjugates were successfully incorporated into the PVC/polyacrylate. These MWCNT/PANI conjugates doped were exposed to different concentrations of ethylene gas to examine their feasibility for ethylene detection.

Fluorine-Free Superhydrophobic Coating with Antibiofilm Properties Based on Pickering Emulsion Templating.

This study presents antibiofilm coating formulations based on Pickering emulsion templating. The coating contains no bioactive material because its antibiofilm properties stem from passive mechanisms that derive solely from the superhydrophobic nature of the coating. Moreover, unlike most of the superhydrophobic formulations, our system is fluorine-free, thus making the method eminently suitable for food and medical applications. The coating formulation is based on water in toluene or xylene emulsions that are stabilized using commercial hydrophobic silica, with polydimethylsiloxane (PDMS) dissolved in toluene or xylene. The structure of the emulsions and their stability was characterized by confocal microscopy and cryogenic-scanning electron microscopy (cryo-SEM). The most stable emulsions are applied on polypropylene (PP) surfaces and dried in an oven to form PDMS/silica coatings in a process called emulsion templating. The structure of the resulting coatings was investigated by atomic force microscopy (AFM) and SEM. The surface of the coatings shows a honeycomb-like structure that exhibits a combination of micron-scale and nanoscale roughness, which endows it with its superhydrophobic properties. After tuning, the superhydrophobic properties of the coatings demonstrated highly efficient passive antibiofilm activity. In vitro antibiofilm trials with E. coli indicate that the coatings reduced the biofilm accumulation by 83% in the xylene-water-based surfaces and by 59% in the case of toluene-water-based surfaces.

Single cell encapsulation in a Pickering emulsion stabilized by TiO2 nanoparticles provides protection against UV radiation for a biopesticide.

A new formulation for biological pest control with significant UV protection capability has been developed in this research. The formulation is based on individual encapsulation of fungal conidia in an oil/water Pickering emulsion. The droplets size of the emulsions was tuned to meet the demands of single conidia encapsulation in the oil droplets. The emulsions are stabilized by amine-functionalized TiO2 (titania) nanoparticles (NPs). The droplet size, stability, and structure of the emulsions were investigated at different TiO2 contents and oil/water phase ratios. Most of the emulsions remained stable for 6 months. The structural properties of the Pickering emulsions were characterized by confocal microscopy and high-resolution cryogenic scanning electron microscopy (cryo-HRSEM). The presence of the TiO2 particles at the interface was confirmed by both confocal microscopy and cryo-HRSEM. Metarhizium brunneum-7 (Mb7) conidia were added to the emulsions. The successful encapsulation of individual conidia in the oil droplets was confirmed by confocal microscopy. The individual encapsulation of the conidia in the emulsions was significantly improved by dispersing the conidia in a 0.02 % Triton X-100 solution prior to emulsification. In addition, the bioassay results have shown, that exposure of the encapsulated conidia to natural UV light did not change their germination rates, however, the unprotected conidia demonstrated a dramatic decrease in their germination rates. These results confirm the UV protection capability of the studied emulsions.

Not Only a Formulation: The Effects of Pickering Emulsion on the Entomopathogenic Action of Metarhizium brunneum.

Growing global population and environmental concerns necessitate the transition from chemical to eco-friendly pest management. Entomopathogenic fungi (EPF) are rising candidates for this task due to their ease of growing, broad host range and unique disease process, allowing EPF to infect hosts directly through its cuticle. However, EPF's requirement for high humidity negates their integration into conventional agriculture. To mitigate this problem, we formulated Metarhizium brunneum conidia in an oil-in-water Pickering emulsion. Conidia in aqueous and emulsion formulations were sprayed on Ricinus communis leaves, and Spodoptera littoralis larvae were introduced under low or high humidity. The following were examined: conidial dispersion on leaf, larval mortality, conidial acquisition by larvae, effects on larval growth and feeding, and dynamic of disease progression. Emulsion was found to disperse conidia more efficiently and caused two-fold more adhesion of conidia to host cuticle. Mortality from conidia in emulsion was significantly higher than other treatments reaching 86.5% under high humidity. Emulsion was also found to significantly reduce larval growth and feeding, while conferring faster fungal growth in-host. Results suggest that a Pickering emulsion is able to improve physical interactions between the conidia and their surroundings, while weakening the host through a plethora of mechanisms, increasing the chance of an acute infection.

In situ Fabrication of Multi-Walled Carbon Nanotubes/Silica Hybrid Colloidosomes by Pickering Emulsion Templating Using Trialkoxysilanes of Opposite Polarity.

A simple and effective way to prepare multi-walled carbon nanotubes (MWNT)//silica hybrid microcapsules (colloidosomes) is presented. These microcapsules have been generated by emulsion templating in a biphasic oil-in-water (o/w) system. Two trialkoxysilanes of complementary polarity, (3-aminopropyl)triethoxysilane (APTES) and dodecyltriethoxysilane (DTES), were used to chemically immobilize the silica nanoparticles at the o/w interface and stabilize the as-generated Pickering emulsions. The effects of varying the o/w ratio and the concentration of the added solids on the type of emulsion formed, the oil droplet size, as well as the emulsion stability have been investigated. The emulsion phase fraction was dependent on the silica content while the droplet size increased with increasing oil volume percentage. A solid shell emerged around the oil droplets from copolymerization between silane monomers. The thickness of the resulting shells was several hundreds of nm. Although MWNTs and silica nanoparticles both were co-assembled at the o/w interface, silica has shown to be the sole stabilizer, with APTES being crucial for the formation of the shell structure. Drop-casting of the emulsion and air-drying led to hierarchical open porous MWNT-silica nanocomposites. These new structures are promising as electrically conductive thin films for variety of applications, such as electro-optics, encapsulation, or chemical sensing.

Single Cell Encapsulation via Pickering Emulsion for Biopesticide Applications.

A new approach for single cell microencapsulation in an oil-in-water (o/w) Pickering emulsion is presented. The water/paraffin emulsions were stabilized by amine-functionalized silica nanoparticles. The droplet size of the emulsions was highly tunable, and ranged from 1 to 30 µm in diameter. The controllable droplet size along with the high colloidal stability of the Pickering emulsionswas harnessed to obtain single cell microencapsulation. Successful encapsulation of the conidia entomopathogenic fungus Metarhizium brunneum by the studied Pickering emulsions was confirmed via confocal laser scanning microscopy. The resulting systems were implemented to develop a novel biopesticide formulation for arthropod pest control. The conidia incorporated in the emulsions were applied to Ricinus communis leaves by spray assay. After drying of the emulsion, a silica-based honeycomb-like structure with an ordered hierarchical porosity is formed. This structure preserves the individual cell encapsulation. The successful single cell encapsulation has led to a high distribution of conidia cells on the leaves. The Pickering emulsion-based formulation exhibited significantly higher pest control activity against Spodoptera littoralis larvae compared to the control systems, thus making it a promising, cost-effective, innovative approach for tackling the pest control challenge.

A Robust Fabrication Method for Amphiphilic Janus Particles via Immobilization on Polycarbonate Microspheres.

Immobilizing particles on beads, fibers, or filaments, when only one side is exposed to the reaction medium and therefore can be selectively functionalized, is a scalable and easy to control strategy for the fabrication of amphiphilic Janus particles. Here we describe a new, robust method for the fabrication of amphiphilic Janus particles based on immobilization of polymethylsilsesquioxane (PMSQ) particles on polycarbonate (PC), a high impact-resistance polymer with superior mechanical properties. The immobilization of the particles on the PC microspores is performed via inverse solvent displacement method. PMSQ particles are added to a PC solution in tetrahydrofuran (THF), a good solvent for PC. The solution is then precipitated by the introduction of aqueous surfactant solution (antisolvent for PC) under an ultrasonic field. It is important to note that THF and water are miscible and do not form emulsion. During precipitation, PMSQ particles are assembled onto the surface of the PC spherical precipitates/microspheres. The exposed hemispheres of the PMSQ particles are then selectively silanized by (3-Aminopropyl)triethoxysilane (APTES) to introduce amine groups on their surface. To increase the polarity of the functionalized hemispheres, the amine groups are further modified to introduce carboxyl groups. SEM characterization confirms the fine embedment of PMSQ particles onto the PC microspheres. Covalent attachment of silica nanoparticles (NPs) to the functionalized hemispheres of the resulting particles along with fluorescent confocal microscopy conclusively prove the successful fabrication of amphiphilic Janus particles. The immobilization of particles onto highly rigid polymeric microspheres such as PC may pave the way for the development of a robust fabrication procedure with high resistance to temperature fluctuations and harsh mixing conditions that can arise during preparation. This method can be implemented toward a large variety of other synthetic commercial polymers such as polyamide, polyether sulfones, Polyether, ether ketone, or similar.

Application of Surface Click Reactions to Localized Surface Plasmon Resonance (LSPR) Biosensing.

Localized surface plasmon resonance (LSPR) spectroscopy is an effective tool for sensitive, affordable, and label-free biosensing. LSPR transducers based on nanoparticulate Au films have been applied to biosensing of receptor-analyte interactions, employing primarily thiolated receptors for constructing biorecognition interfaces on nanostructured Au surfaces. This popular method suffers from a major drawback, that is, the need to prepare a thiolated receptor for each system used, which is typically synthetically complex and time-consuming. Herein, we present an alternative approach based on the click reaction between azide and terminal alkyne, which avoids the need to synthesize thiol-derivatized receptors and is applicable to the heterogeneous morphology of LSPR transducers. The receptors are tethered with an alkyne group, which is considerably simpler than thiolation, while producing a stable product. The transducer surface is modified with a layer of a commercial long-chain thiol-azide molecule, then clicked with an alkyne-dertivatized receptor to produce the biorecognition interface. This method is employed for immobilization of four different alkyne-bearing receptor molecules on Au nano-island film based LSPR transducers, followed by testing of their performance in biorecognition of specific analytes using LSPR and FTIR spectroscopies. The results establish the usefulness of click chemistry for the preparation of biorecognition interfaces on nanostructured LSPR transducers.

Direct fluorescence detection of VirE2 secretion by Agrobacterium tumefaciens.

VirE2 is a ssDNA binding protein essential for virulence in Agrobacterium tumefaciens. A tetracysteine mutant (VirE2-TC) was prepared for in vitro and in vivo fluorescence imaging based on the ReAsH reagent. VirE2-TC was found to be biochemically active as it binds both ssDNA and the acidic secretion chaperone VirE1. It was also biologically functional in complementing virE2 null strains transforming Arabidopsis thaliana roots and Nicotiana tabacum leaves. In vitro experiments demonstrated a two-color fluorescent complex using VirE2-TC/ReAsH and Alexa Fluor 488 labeled ssDNA. In vivo, fluorescent VirE2-TC/ReAsH was detected in bacteria and in plant cells at time frames relevant to transformation.

Effect of a single amino acid substitution in the NLS domain of Tomato yellow leaf curl virus-Israel (TYLCV-IL) capsid protein (CP) on its activity and on the virus life cycle.

The capsid protein (CP) of Tomato yellow leaf curl virus-Israel (TYLCV-IL), encoded by the v1 gene, is the only known component of the viral capsid. Three point mutations introduced into the conserved NLS region of the CP were investigated. One mutant, in which the Arg at position 19 was converted to Leu, had the most significant effect on the CP-CP homotypic interaction as well as on CP's interaction with its nuclear receptor karyopherin α1 and with the protein GroEL. The latter has been suggested to protect the virions in the insect vector hemolymph. These effects were first observed by yeast two-hybrid assay and then confirmed in tobacco protoplasts by measuring fluorescence resonance energy transfer (FRET) between YFP- and CFP-tagged proteins. Most importantly, when the point mutation converting Arg 19 to Leu was introduced into the full-length TYLCV genome, it disrupted its ability to cause symptoms.