Plant Membrane-On-A-Chip: A Platform for Studying Plant Membrane Proteins and Lipids.

The cell plasma membrane is a two-dimensional, fluid mosaic material composed of lipids and proteins that create a semipermeable barrier defining the cell from its environment. Compared with soluble proteins, the methodologies for the structural and functional characterization of membrane proteins are challenging. An emerging tool for studies of membrane proteins in mammalian systems is a "plasma membrane on a chip," also known as a supported lipid bilayer. Here, we create the "plant-membrane-on-a-chip,″ a supported bilayer made from the plant plasma membranes of Arabidopsis thaliana, Nicotiana benthamiana, or Zea mays. Membrane vesicles from protoplasts containing transgenic membrane proteins and their native lipids were incorporated into supported membranes in a defined orientation. Membrane vesicles fuse and orient systematically, where the cytoplasmic side of the membrane proteins faces the chip surface and constituents maintain mobility within the membrane plane. We use plant-membrane-on-a-chip to perform fluorescent imaging to examine protein-protein interactions and determine the protein subunit stoichiometry of FLOTILLINs. We report here that like the mammalian FLOTILLINs, FLOTILLINs expressed in Arabidopsis form a tetrameric complex in the plasma membrane. This plant-membrane-on-a-chip approach opens avenues to studies of membrane properties of plants, transport phenomena, biophysical processes, and protein-protein and protein-lipid interactions in a convenient, cell-free platform.

Distinguishing cells using electro-acoustic spinning.

Many diseases, including cancer and covid, result in altered mechanical and electric properties of the affected cells. These changes were proposed as disease markers. Current methods to characterize such changes either provide very limited information on many cells or have extremely low throughput. We introduce electro-acoustic spinning (EAS). Cells were found to spin in combined non-rotating AC electric and acoustic fields. The rotation velocity in EAS depends critically on a cell's electrical and mechanical properties. In contrast to existing methods, the rotation is uniform in the field of view and hundreds of cells can be characterized simultaneously. We demonstrate that EAS can distinguish cells with only minor differences in electric and mechanical properties, including differences in age or the number of passages.

Grafting of Cyclodextrin to Theranostic Nanoparticles Improves Blood-Brain Barrier Model Crossing.

Core-shell superparamagnetic iron oxide nanoparticles hold great promise as a theranostic platform in biological systems. Herein, we report the biological effect of multifunctional cyclodextrin-appended SPIONs (CySPION) in mutant Npc1-deficient CHO cells compared to their wild type counterparts. CySPIONs show negligible cytotoxicity while they are strongly endocytosed and localized in the lysosomal compartment. Through their bespoke pH-sensitive chemistry, these nanoparticles release appended monomeric cyclodextrins to mobilize over-accumulated cholesterol and eject it outside the cells. CySPIONs show a high rate of transport across blood-brain barrier models, indicating their promise as a therapeutic approach for cholesterol-impaired diseases affecting the brain.

Rolling Circle Amplification Tailored for Plasmonic Biosensors: From Ensemble to Single-Molecule Detection.

We report on the tailoring of rolling circle amplification (RCA) for affinity biosensors relying on the optical probing of their surface with confined surface plasmon field. Affinity capture of the target analyte at the metallic sensor surface (e.g., by using immunoassays) is followed by the RCA step for subsequent readout based on increased refractive index (surface plasmon resonance, SPR) or RCA-incorporated high number of fluorophores (in surface plasmon-enhanced fluorescence, PEF). By combining SPR and PEF methods, this work investigates the impact of the conformation of long RCA-generated single-stranded DNA (ssDNA) chains to the plasmonic sensor response enhancement. In order to confine the RCA reaction within the evanescent surface plasmon field and hence maximize the sensor response, an interface carrying analyte-capturing molecules and additional guiding ssDNA strands (complementary to the repeating segments of RCA-generated chains) is developed. When using the circular padlock probe as a model target analyte, the PEF readout shows that the reported RCA implementation improves the limit of detection (LOD) from 13 pM to high femtomolar concentration when compared to direct labeling. The respective enhancement factor is of about 2 orders of magnitude, which agrees with the maximum number of fluorophore emitters attached to the RCA chain that is folded in the evanescent surface plasmon field by the developed biointerface. Moreover, the RCA allows facile visualizing of individual binding events by fluorescence microscopy, which enables direct counting of captured molecules. This approach offers a versatile route toward a fast digital readout format of single-molecule detection with further reduced LOD.

Interplay between biofilm microenvironment and pathogenicity of Pseudomonas aeruginosa in cystic fibrosis lung chronic infection.

Pseudomonas aeruginosa (PA) is a highly, if not the most, versatile microorganism capable of colonizing diverse environments. One of the niches in which PA is able to thrive is the lung of cystic fibrosis (CF) patients. Due to a genetic aberration, the lungs of CF-affected patients exhibit impaired functions, rendering them highly susceptible to bacterial colonization. Once PA attaches to the epithelial surface and transitions to a mucoid phenotype, the infection becomes chronic, and antibiotic treatments become inefficient. Due to the high number of affected people and the severity of this infection, CF-chronic infection is a well-documented disease. Still, numerous aspects of PA CF infection remain unclear. The scientific reports published over the last decades have stressed how PA can adapt to CF microenvironmental conditions and how its surrounding matrix of extracellular polymeric substances (EPS) plays a key role in its pathogenicity. In this context, it is of paramount interest to present the nature of the EPS together with the local CF-biofilm microenvironment. We review how the PA biofilm microenvironment interacts with drugs to contribute to the pathogenicity of CF-lung infection. Understanding why so many drugs are inefficient in treating CF chronic infection while effectively treating planktonic PA is essential to devising better therapeutic targets and drug formulations.

Cellulosic biofilm formation of Komagataeibacter in kombucha at oil-water interfaces.

Bacteria forming biofilms at oil-water interfaces have diverse metabolism, they use hydrocarbons as a carbon and energy source. Kombucha is a fermented drink obtained from a complex symbiotic culture of bacteria and yeast, where acetic acid bacteria present in kombucha use sugars as a carbon source to produce cellulosic biofilms. We hypothesize that Komagataeibacteraceae in kombucha can adsorb to and use hydrocarbons as the sole energy source to produce cellulosic biofilms. Hence we characterized a kombucha culture, studied bacterial adsorption and cellulosic biofilm formation of kombucha at the n-decane or mineral oil-kombucha suspension interface. The cellulosic biofilms were imaged using fluorescence microscopy and cryo-scanning electron microscopy, and their time-dependent rheology was measured. Komagataeibacter, the dominant bacterial genus in the kombucha culture, produced cellulosic biofilms with reduced cellulose biomass yield at the oil-kombucha suspension interfaces compared to at the air-kombucha suspension interface. The presence of biosurfactants in the supernatant secreted by the kombucha microbes led to a larger and faster decrease in the interfacial tension on both oil types, leading to the formation of stable and elastic biofilm membranes. The difference in interfacial tension reduction was insignificant already after 2 h of biofilm formation at the mineral oil-kombucha suspension interface compared to kombucha microbes resuspended without biosurfactants but persisted for longer than 24 h in contact with n-decane. We also demonstrate that Komagataeibacter in kombucha can produce elastic cellulosic biofilms using hydrocarbons from the oil interface as the sole source of carbon and energy. Thus Komagataeibacter and kombucha shows the potential of this system for producing valued bacterial cellulose through remediation of hydrocarbon waste.

A microfluidic valve with bubble trap and zero dead volume.

We present a technique to swiftly change the contents of a small sample chamber using only a few times the chamber volume. Our design has no dead volume and functions as a manifold that minimizes mixing between consecutive liquids at one inlet. Thereby, it is ideal for minimizing sample consumption. In addition, our fluidic circuit works as an efficient bubble trap. These properties make our design an exciting alternative to standard solutions using multiple valves and junctions.

Modifying superparamagnetic iron oxide and silica nanoparticles surfaces for efficient (MA)LDI-MS analyses of peptides and proteins.

RATIONALE: Surface functionalization is considered to be the foundation for developing nanomaterial applications in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analyses. However, the surface properties of nanostructures can influence their interaction with the analyte and consequently the mass data. In the present study, functionalized nanoparticles (NPs) were used for MALDI-MS and laser desorption/ionization mass spectrometry (LDI-MS) experiments in order to evaluate the effect of the surface properties of NPs on tailoring the intensity of mass signals. METHODS: Regarding the LDI-MS analyses, the surface of superparamagnetic iron oxide nanoparticles (SPIONs) was coated with nitrosonium tetrafluoroborate, citric acid, nitrodopamine, and gallic acid. Additionally, the SPIONs were applied as a matrix to analyze three small peptides. In the MALDI-MS analyses, silica NPs were selected as co-matrix and functionalized with cysteine, sulfobetaine, and amine alkoxysilanes. Then, the silica NPs were utilized as additives in the MALDI-MS samples of four proteins in a mass range between ~2000 and 60,000 Da. RESULTS: The results of LDI-MS analyses demonstrated more than one order enhancement in the signal intensity of analytes based on the amount of electrostatic interaction and laser energy absorption by the surface ligands. However, those of MALDI-MS experiments indicated a significant signal improvement when achieving the colloidal stability of silica NPs in the matrix solution. CONCLUSIONS: Based on the results, the surface properties of NPs affected the (MA)LDI-MS analyses indispensably. Finally, the functionalization of SPIONs represented a new model for the future development of NPs with both affinity and enhanced ionization abilities in mass spectrometry.

Cyclodextrin-Appended Superparamagnetic Iron Oxide Nanoparticles as Cholesterol-Mopping Agents.

Cholesterol plays a crucial role in major cardiovascular and neurodegenerative diseases, including Alzheimer's disease and rare genetic disorders showing altered cholesterol metabolism. Cyclodextrins (CDs) have shown promising therapeutic efficacy based on their capacity to sequester and mobilise cholesterol. However, the administration of monomeric CDs suffers from several drawbacks due to their lack of specificity and poor pharmacokinetics. We present core-shell superparamagnetic iron oxide nanoparticles (SPIONs) functionalised with CDs appended to poly (2-methyl-2-oxazoline) polymers grafted in a dense brush to the iron oxide core. The CD-decorated nanoparticles (CySPIONs) are designed so that the macrocycle is specifically cleaved off the nanoparticle's shell at a slightly acidic pH. In the intended use, free monomeric CDs will then mobilise cholesterol out of the lysosome to the cytosol and beyond through the formation of an inclusion complex. Hence, its suitability as a therapeutic platform to remove cholesterol in the lysosomal compartment. Synthesis and full characterization of the polymer as well as of the core-shell SPION are presented. Cholesterol-binding activity is shown through an enzymatic assay.

Method for High-Yield Hydrothermal Growth of Silica Shells on Nanoparticles.

Coating processes are commonly used in materials science to protect a core or modify material properties. We describe a hydrothermal coating process using TEOS (tetraethyl orthosilicate), a widely used precursor for silica coatings, on three representative template materials (carbon nanotubes, silica, and polystyrene nanoparticles) with different properties and shapes. We compare the efficiency of previously published protocols for silica coatings at room temperature and atmospheric pressure with the hydrothermal process at 160 °C and 3 bar. The hydrothermal method achieves higher yields and thicker silica coatings with the same amount of precursor when compared to the conventional way, thus offering higher effectiveness. Furthermore, the hydrothermal coating process yields more homogeneous shells with a higher density, making hydrothermal coating the method of choice when mechanical integrity and low permeability of the coating are required.

Understanding the Photochemical Properties of Polythiophene Polyelectrolyte Soft Aggregates with Sodium Dodecyl Sulfate for Antimicrobial Activity.

The threat of antibiotic-resistant bacteria is an ever-increasing problem in public health. In this report, we examine the photochemical properties with a proof-of-principle biocidal assay for a novel series of regio-regular imidazolium derivative poly-(3-hexylthiophene)/sodium dodecyl sulfate (P3HT-Im/SDS) materials from ultrafast sub-ps dynamics to µs generation of reactive oxygen species (ROS) and 30 min biocidal reactivity with Escherichia coli (E. coli). This broad series encompassing pure P3HT-Im to cationic, neutral, and anionic P3HT-Im/SDS materials are all interrogated by a variety of techniques to characterize the physical material structure, electronic structure, and antimicrobial activity. Our results show that SDS complexation with P3HT-Im results in aggregate materials with reduced ROS generation and light-induced anti-microbial activity. However, our characterization reveals that the presence of non-aggregated or lightly SDS-covered polymer segments is still capable of ROS generation. Full encapsulation of the P3HT-Im polymer completely deactivates the light killing pathway. High SDS concentrations, near and above critical micelle concentration, further deactivate all anti-microbial activity (light and dark) even though the P3HT-Im regains its electronic properties to generate ROS.

Hybrid lipopolymer vesicle drug delivery and release systems.

Hybrid lipopolymer vesicles are membrane vesicles that can be self-assembled on both the micro- and nano-scale. On the nanoscale, they are potential novel smart materials for drug delivery systems that could combine the relative strengths of liposome and polymersome drug delivery systems without their respective weaknesses. However, little is known about their properties and how they could be tailored. Currently, most methods of investigation are limited to the microscale. Here we provide a brief review on hybrid vesicle systems with a specific focus on recent developments demonstrating that nanoscale hybrid vesicles have different properties from their macroscale counterparts.

DNA Polyelectrolyte Multilayer Coatings Are Antifouling and Promote Mammalian Cell Adhesion.

The ability of bacteria to adhere to and form biofilms on implant surfaces is the primary cause of implant failure. Implant-associated infections are difficult to treat, as the biofilm mode of growth protects microorganisms from the host's immune response and antibiotics. Therefore, modifications of implant surfaces that can prevent or delay bacterial adhesion and biofilm formation are highly desired. In addition, the attachment and spreading of bone cells are required for successful tissue integration in orthopedic and dental applications. We propose that polyanionic DNA with a negatively charged phosphate backbone could provide a dual function to repel bacterial adhesion and support host tissue cell attachment. To this end, we developed polyelectrolyte multilayer coatings using chitosan (CS) and DNA on biomaterial surfaces via a layer-by-layer technique. The assembly of these coatings was characterized. Further, we evaluated staphylococcal adhesion and biofilm growth on the coatings as well as cytotoxicity for osteoblast-like cells (SaOS-2 cells), and we correlated these to the layer structure. The CS-DNA multilayer coatings impaired the biofilm formation of Staphylococcus by ~90% on both PMMA and titanium surfaces. The presence of cationic CS as the top layer did not hinder the bacteria-repelling property of the DNA in the coating. The CS-DNA multilayer coatings demonstrated no cytotoxic effect on SaOS-2 cells. Thus, DNA polyelectrolyte multilayer coatings could reduce infection risk while promoting host tissue cell attachment on medical implants.

In Situ Monitoring of Rolling Circle Amplification on a Solid Support by Surface Plasmon Resonance and Optical Waveguide Spectroscopy.

The growth of surface-attached single-stranded deoxyribonucleic acid (ssDNA) chains is monitored in situ using an evanescent wave optical biosensor that combines surface plasmon resonance (SPR) and optical waveguide spectroscopy (OWS). The "grafting-from" growth of ssDNA chains is facilitated by rolling circle amplification (RCA), and the gradual prolongation of ssDNA chains anchored to a gold sensor surface is optically tracked in time. At a sufficient density of the polymer chains, the ssDNA takes on a brush architecture with a thickness exceeding 10 µm, supporting a spectrum of guided optical waves traveling along the metallic sensor surface. The simultaneous probing of this interface with the confined optical field of surface plasmons and additional more delocalized dielectric optical waveguide modes enables accurate in situ measurement of the ssDNA brush thickness, polymer volume content, and density gradients. We report for the first time on the utilization of the SPR/OWS technique for the measurement of the RCA speed on a solid surface that can be compared to that in bulk solutions. In addition, the control of ssDNA brush properties by changing the grafting density and ionic strength and post-modification via affinity reaction with complementary short ssDNA staples is discussed. These observations may provide important leads for tailoring RCA toward sensitive and rapid assays in affinity-based biosensors.

Thermoresponsive Nanoparticles with Cyclic-Polymer-Grafted Shells Are More Stable than with Linear-Polymer-Grafted Shells: Effect of Polymer Topology, Molecular Weight, and Core Size.

Polymer brush-grafted superparamagnetic iron oxide nanoparticles can change their aggregation state in response to temperature and are potential smart materials for many applications. Recently, the shell morphology imposed by grafting to a nanoparticle core was shown to strongly influence the thermoresponsiveness through a coupling of intrashell solubility transitions and nanoparticle aggregation. We investigate how a change from linear to cyclic polymer topology affects the thermoresponsiveness of poly(2-isopropyl-2-oxazoline) brush-grafted superparamagnetic iron oxide nanoparticles. Linear and cyclic polymers with three different molecular weights (7, 18, and 24.5 kg mol-1) on two different core sizes (3.7 and 9.2 nm) and as free polymer were investigated. We observed the critical flocculation temperature (CFT) during temperature cycling dynamic light scattering experiments, the critical solution temperature (CST), and the transition enthalpy per monomer during differential scanning calorimetry measurements. When all conditions are identical, cyclic polymers increase the colloidal stability and the critical flocculation temperature compared to their linear counterparts. Furthermore, the cyclic polymer shows only one uniform transition, while we observe multiple transitions for the linear polymer shells. We link the single transition and higher colloidal stability to the absence in cyclic PiPrOx shells of a dilute outer part where the particle shells can interdigitate.

Morpholinium-based ionic liquids show antimicrobial activity against clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a multi-drug resistant (MDR) pathogen. It is classified by WHO as one of the most life-threatening pathogens causing nosocomial infections. Some of its clinical isolates and their subpopulations show high persistence to many antibiotics that are recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Thus, there is a need for non-traditional classes of antibiotics to fight the increasing threat of MDR P. aeruginosa. Ionic liquids (IL) are one such promising class of novel antibiotics. We selected four strains of P. aeruginosa and studied the growth inhibition and other effects of 12 different ILs. We used the well-characterized P. aeruginosa PAO1 (ATCC 15692) as model strain and compared it to three other isolates from chronic lung infection (LES B58), skin burn infection (UCBPP-PA14) and keratitis infection (39016), respectively. The ILs consisted of either 4,4-didecylmorpholinium [Dec2Mor]+ or 4-decyl-4-ethylmorpholinium [DecEtMor]+ cations combined with different anions. We found that the ILs with 4,4-didecylmorpholinium [Dec2Mor]+ cations most effectively inhibited bacterial growth as well as reduced strain fitness and virulence factor production. Our results indicate that these ILs could be used to treat P. aeruginosa infections.

Synthesis of short-range ordered aluminosilicates at ambient conditions.

We report here on structure-related aggregation effects of short-range ordered aluminosilicates (SROAS) that have to be considered in the development of synthesis protocols and may be relevant for the properties of SROAS in the environment. We synthesized SROAS of variable composition by neutralizing aqueous aluminium chloride with sodium orthosilicate at ambient temperature and pressure. We determined elemental composition, visualized morphology by microscopic techniques, and resolved mineral structure by solid-state 29Si and 27Al nuclear magnetic resonance and Fourier-transform infrared spectroscopy. Nitrogen sorption revealed substantial surface loss of Al-rich SROAS that resembled proto-imogolite formed in soils and sediments due to aggregation upon freezing. The effect was less pronounced in Si-rich SROAS, indicating a structure-dependent effect on spatial arrangement of mass at the submicron scale. Cryomilling efficiently fractured aggregates but did not change the magnitude of specific surface area. Since accessibility of surface functional groups is a prerequisite for sequestration of substances, elucidating physical and chemical processes of aggregation as a function of composition and crystallinity may improve our understanding of the reactivity of SROAS in the environment.

Theoretical and Experimental Design of Heavy Metal-Mopping Magnetic Nanoparticles.

Herein, we show a comprehensive experimental, theoretical, and computational study aimed at designing macromolecules able to adsorb a cargo at the nanoscale. Specifically, we focus on the adsorption properties of star diblock copolymers, i.e., macromolecules made by a number f of H-T diblock copolymer arms tethered on a central core; the H monomeric heads, which are closer to the tethering point, are attractive toward a specific target, while the T monomeric tails are neutral to the cargo. Experimentally, we exploited the adaptability of poly(2-oxazoline)s (POxs) to realize block copolymer-coated nanoparticles with a proper functionalization able to interact with heavy metals and show or exhibit a thermoresponsive behavior in aqueous solution. We here present the synthesis and analysis of the properties of a high molecular mass block copolymer featured by (i) a polar side chain, capable of exploiting electrostatic and hydrophilic interaction with a predetermined cargo, and (ii) a thermoresponsive scaffold, able to change the interaction with the media by tuning the temperature. Afterward, the obtained polymers were grafted onto iron oxide nanoparticles and the thermoresponsive properties were investigated. Through isothermal titration calorimetry, we then analyzed the adsorption properties of the synthesized superparamagnetic nanoparticles for heavy metal ions in aqueous solution. Additionally, we use a combination of scaling theories and simulations to link equilibrium properties of the system to a prediction of the loading properties as a function of size ratio and effective interactions between the considered species. The comparison between experimental results on adsorption and theoretical prediction validates the whole design process.

Polymer Brush-Grafted Nanoparticles Preferentially Interact with Opsonins and Albumin.

Nanoparticles find increasing applications in life science and biomedicine. The fate of nanoparticles in a biological system is determined by their protein corona, as remodeling of their surface properties through protein adsorption triggers specific recognition such as cell uptake and immune system clearance and nonspecific processes such as aggregation and precipitation. The corona is a result of nanoparticle-protein and protein-protein interactions and is influenced by particle design. The state-of-the-art design of biomedical nanoparticles is the core-shell structure exemplified by superparamagnetic iron oxide nanoparticles (SPIONs) grafted with dense, well-hydrated polymer shells used for biomedical magnetic imaging and therapy. Densely grafted polymer chains form a polymer brush, yielding a highly repulsive barrier to the formation of a protein corona via nonspecific particle-protein interactions. However, recent studies showed that the abundant blood serum protein albumin interacts with dense polymer brush-grafted SPIONs. Herein, we use isothermal titration calorimetry to characterize the nonspecific interactions between human serum albumin, human serum immunoglobulin G, human transferrin, and hen egg lysozyme with monodisperse poly(2-alkyl-2-oxazoline)-grafted SPIONs with different grafting densities and core sizes. These particles show similar protein interactions despite their different "stealth" capabilities in cell culture. The SPIONs resist attractive interactions with lysozymes and transferrins, but they both show a significant exothermic enthalpic and low exothermic entropic interaction with low stoichiometry for albumin and immunoglobulin G. Our results highlight that protein size, flexibility, and charge are important to predict protein corona formation on polymer brush-stabilized nanoparticles.