High temperature mass detection using a carbon nanotube bilayer modified quartz crystal microbalance as a GC detector.

A small, portable gas chromatograph (GC) was assembled for the trace detection of controlled substances using a novel quartz crystal microbalance sensor (QCM). The QCM crystal surface was modified with a variety of sorption materials to increase adsorption thereby amplifying mass detection. Single polymer thin film coatings increased the QCM response by 1-2 orders of magnitude, while operating at over 100 °C. Adding a layer of carbonaceous nanomaterial (graphene or carbon nanotubes) above such a film dramatically increased sensitivity by up to 3 orders of magnitude compared to uncoated crystals. Separation and detection of submicrogram quantities of controlled substances was carried out within minutes by employing a GC column and detector temperature ramp up to 220 °C. An additional 10-fold enhancement in sensitivity was achieved by mechanical abrasion of the sample swabs used in the sample introduction process. This study demonstrated a novel use of a polymer composite modified QCM as a chemical sensor at high temperatures.

Rescue and characterization of the first West African Marburg virus 2021 from Guinea.

Marburg virus (MARV) is a causative agent of a severe hemorrhagic fever with high fatality rates endemic in central Africa. Current outbreaks of MARV in Equatorial Guinea and Tanzania underline the relevance of MARV as a public health emergency pathogen. In 2021, the first known human MARV case was confirmed in Guinea, West Africa. Since no infectious virus could be isolated from that fatal case in 2021, we generated recombinant (rec) MARV Guinea by reverse genetics in order to study and characterize this new MARV, which occurred in West Africa for the first time, in terms of its growth properties, detection by antibodies, and therapeutic potential compared to known MARV strains. Our results showed a solid viral replication of recMARV Guinea in human, bat, and monkey cell lines in comparison to other known MARV strains. We further demonstrated that replication of recMARV Guinea in cells can be inhibited by the nucleoside analogue remdesivir. Taken together, we could successfully reconstitute de novo the first West African MARV from Guinea showing similar replication kinetics in cells compared to other central African MARV strains. Our reverse genetics approach has proven successful in characterizing emerging viruses, especially when virus isolates are missing and viral genome sequences are incomplete.

Antigen modifications improve nucleoside-modified mRNA-based influenza virus vaccines in mice.

Nucleoside-modified, lipid nanoparticle-encapsulated mRNAs have recently emerged as suitable vaccines for influenza viruses and other pathogens in part because the platform allows delivery of multiple antigens in a single immunization. mRNA vaccines allow for easy antigen modification, enabling rapid iterative design. We studied protein modifications such as mutating functional sites, changing secretion potential, and altering protein conformation, which could improve the safety and/or potency of mRNA-based influenza virus vaccines. Mice were vaccinated intradermally with wild-type or mutant constructs of influenza virus hemagglutinin (HA), neuraminidase (NA), matrix protein 2 (M2), nucleoprotein (NP), or matrix protein 1 (M1). Membrane-bound HA constructs elicited more potent and protective antibody responses than secreted forms. Altering the catalytic site of NA to reduce enzymatic activity decreased reactogenicity while protective immunity was maintained. Disruption of M2 ion channel activity improved immunogenicity and protective efficacy. A comparison of internal proteins NP and M1 revealed the superiority of NP in conferring protection from influenza virus challenge. These findings support the use of the nucleoside-modified mRNA platform for guided antigen design for influenza virus with extension to other pathogens.

The deformation and adhesion of randomly rough and patterned surfaces.

Using a surface forces apparatus (SFA) and an atomic force microscope (AFM) we have studied the effects of surface roughness (root-mean-square (RMS) roughness between 0.3 and 220 nm) on the "contact mechanics", which describes the deformations and loading and unloading adhesion forces, of various polymeric surfaces. For randomly rough, moderately stiff, elastomeric surfaces, the force-distance curves on approach and separation are nearly reversible and almost perfectly exponentially repulsive, with an adhesion on separation that decreases only slightly with increasing RMS. Additionally, the magnitude of the preload force is seen to play a large role in determining the measured adhesion. The exponential repulsion likely arises from the local compressions (fine-grained nano- or submicron-scale deformations) of the surface asperities. The resulting characteristic decay lengths of the repulsion scale with the RMS roughness and correlate very well with a simple finite element method (FEM) analysis based on actual AFM topographical images of the surfaces. For "patterned" surfaces, with a nonrandom terraced structure, no similar exponential repulsion is observed, suggesting that asperity height variability or random roughness is required for the exponential behavior. However, the adhesion force or energy between two "patterned" surfaces fell off dramatically and roughly exponentially as the RMS increased, likely owing to a significant decrease in the contact area which in turn determines their adhesion. For both types of rough surfaces, random and patterned, the coarse-grained (global, meso- or macroscopic) deformations of the initially curved surfaces appear to be Hertzian.

Lubrication and wear properties of grafted polyelectrolytes, hyaluronan and hylan, measured in the surface forces apparatus.

Hyaluronan is believed to have an important function in the boundary biolubrication of articular cartilage. Using a Surface Forces Apparatus, we tested the tribological properties of surface bound, rather than "free" hyaluronan. The grafting process of the polyelectrolyte included either a biological route via an HA-binding protein or a chemical reaction to covalently bind the polymer to a lipid bilayer coated surface. In another reaction, we constructed a surface with covalently grafted hylan (crosslinked hyaluronan). We studied the normal and shear forces between these surfaces. None of the systems demonstrated comparable lubrication to that found between cartilage surfaces except at very low loads. Both grafted hyaluronan and hylan generated coefficients of friction between 0.15 and 0.3. Thus, the polysaccharide, which is a constituent of the lamina splendens (outermost cartilage layer), is not expected to be the responsible molecule for the great lubricity of cartilage; however, it may contribute to the load bearing and wear protection of these surfaces. This was concluded from the results with hylan, where a thin gel layer was sufficient to shield the underlying surfaces from damage even at applied pressures of over 200 atmospheres during shear. Our study shows that a low coefficient of friction is not a requirement for, or necessarily a measure of, wear protection.

Static forces, structure and flow properties of complex fluids in highly confined geometries.

The Surface Forces Apparatus has been successfully used to measure the static and dynamic forces between surfaces across ultra-thin films of water and aqueous electrolyte solutions, and--more recently--polyelectrolyte-coated and articular cartilage surfaces in various solutions including hyaluronan, lubricin, and synovial fluid. The results give new insights into the lubricating action of biological lubricants such as synovial fluid and hyaluronan (a polysaccharide in synovial fluid), and biological surfaces such as phospholipid bilayers and cartilage surfaces. Contrary to earlier indications of long-range water-structuring at biological surfaces, more recent measurements clearly show that the viscosity of physiologically concentrated water (saline) is bulk-like beyond the first 1 or 2 layers from a single surface, and beyond 4-6 layers in thin films between two surfaces (the structure and forces may, however, be affected to larger distances). This implies that most structural, interaction force, and viscosity-related phenomena are determined--not only by the properties of the solvent (water)per se--but also by the surfaces and the water, ions, solutes, and macromolecules (proteins, polymers) exposed or adsorbed at the surfaces and, to a lesser degree, dissolved in the solvent. However, sometimes it is difficult to make a clear differentiation, e.g., one could consider hydration or surface-bound 'structured' water as part of the surface or as part of the intervening water between the two surfaces.

Correlation of AFM and SFA measurements concerning the stability of supported lipid bilayers.

Phospholipid bilayers were studied by means of atomic force microscopy (AFM) and a surface force apparatus (SFA). The stability of the supported bilayers was described by the amount of irregularities in the topography of the membrane by means of AFM and by the occurrence of hemifusion in the SFA, which is an indicator of defective bilayers. The bilayers, composed of lipids having the same headgroup but different chain lengths in the two leaflets, were prepared by Langmuir-Blodgett deposition and transferred at different surface pressures. The topography of the supported bilayers in aqueous solution, as imaged by AFM, revealed an increasing number of defects in the supported lipid membranes with decreased deposition pressure of the outer lipid layer. These defects, which appeared in the form of monolayer and bilayer (self-assembled) thick holes within the membrane, were energetically favorable over an evenly depleted bilayer. We found that the quantity of these defects (holes of