Collection of serum albumin aggregate nanoparticles from human plasma by dielectrophoresis.

Dielectrophoresis (DEP) is a fast and reliable nanoparticle recovery method that utilizes nonuniform electric fields to manipulate particles based on their material composition and size, enabling recovery of biologically-derived nanoparticles from plasma for diagnostic applications. When applying DEP to undiluted human plasma, collection of endogenous albumin proteins was observed at electric field gradients much lower than predicted by theory to collect molecular proteins. To understand this collection, nanoparticle tracking analysis of bovine serum albumin (BSA) dissolved in 0.5× phosphate-buffered saline was performed and showed that albumin spontaneously formed aggregate nanoparticles with a mean diameter of 237 nm. These aggregates experienced a dielectrophoretic force as a function of aggregate radius rather than the diameter of individual protein molecules which contributed to their collection. In high conductance buffer (6.8 mS/cm), DEP was able to move these aggregates into regions of high electric field gradient, and in lower conductance buffer (0.68 mS/cm), these aggregates could be moved into high or low gradient regions depending on the applied frequency. Disruption of BSA aggregates using a nonionic detergent significantly decreased the particle diameter, resulting in decreased dielectrophoretic collection of albumin which increased the collection consistency of particles of interest. These results provide techniques to manipulate albumin aggregates via DEP, which impacts collection of diagnostic biomarkers.

Enhancement of dielectrophoresis-based particle collection from high conducting fluids due to partial electrode insulation.

Dielectrophoresis (DEP) is a successful method to recover nanoparticles from different types of fluid. The DEP force acting on these particles is created by an electrode microarray that produces a nonuniform electric field. To apply DEP to a highly conducting biological fluid, a protective hydrogel coating over the metal electrodes is required to create a barrier between the electrode and the fluid. This protects the electrodes, reduces the electrolysis of water, and allows the electric field to penetrate into the fluid sample. We observed that the protective hydrogel layer can separate from the electrode and form a closed domed structure and that collection of 100 nm polystyrene beads increased when this occurred. To better understand this collection increase, we used COMSOL Multiphysics software to model the electric field in the presence of the dome filled with different materials ranging from low-conducting gas to high conducting phosphate-buffered saline fluids. The results suggest that as the electrical conductivity of the material inside the dome is reduced, the whole dome acts as an insulator which increases electric field intensity at the electrode edge. This increased intensity widens the high-intensity electric field factor zone resulting in increased collection. This informs how dome formation results in increased particle collection and provides insight into how the electric field can be intensified to the increase collection of particles. These results have important applications for increasing the recovery of biologically-derived nanoparticles from undiluted physiological fluids that have high conductance, including the collection of cancer-derived extracellular vesicles from plasma for liquid biopsy applications.

Development of a transient inline spiking system for evaluating virus clearance in continuous bioprocessing-Proof of concept for virus filtration.

The development of continuous/connected bioprocesses requires new approaches for viral clearance validation, both for specific unit operations and for the overall process. In this study, we have developed a transient inline spiking system that can be used to evaluate virus clearance at distinct time points during prolonged operation of continuous bioprocesses. The proof of concept for this system was demonstrated by evaluating the viral clearance for a virus filtration step, both with and without a prefilter upstream of the virus filter. The residence time distribution was evaluated using a previously identified noninteracting fluorescent tracer, while viral clearance was evaluated from measurements of the virus titer in samples obtained downstream of the virus filter. The measured log reduction values (LRV) for ϕX174, minute virus of mice, xenotropic murine leukemia virus, and a noninfectious mock virus particle were all within 0.5 log of those obtained using a traditional batch virus challenge for both model and real-world process streams (LRV between 2.2 and 3.4 for ϕX174 using a single layer of virus filter). The results demonstrate the effectiveness of transient inline spiking to validate the virus clearance capabilities in continuous bioprocessing, an essential element for the adoption of these processes for products made using mammalian cell lines.

Evaluating Protein Fouling on Membranes Patterned by Woven Mesh Fabrics.

Membrane surface patterning is one approach used to mitigate fouling. This study used a combination of flux decline measurements and visualization experiments to evaluate the effectiveness of a microscale herringbone pattern for reducing protein fouling on polyvinylidene fluoride (PVDF) ultrafiltration membranes. Thermal embossing with woven mesh stamps was used for the first time to pattern membranes. Embossing process parameters were studied to identify conditions replicating the mesh patterns with high fidelity and to determine their effect on membrane permeability. Permeability increased or remained constant when patterning at low pressure (≤4.4 MPa) as a result of increased effective surface area; whereas permeability decreased at higher pressures due to surface pore-sealing of the membrane active layer upon compression. Flux decline measurements with dilute protein solutions showed monotonic decreases over time, with lower rates for patterned membranes than as-received membranes. These data were analyzed by the Hermia model to follow the transient nature of fouling. Confocal laser scanning microscopy (CLSM) provided complementary, quantitative, spatiotemporal information about protein deposition on as-received and patterned membrane surfaces. CLSM provided a greater level of detail for the early (pre-monolayer) stage of fouling than could be deduced from flux decline measurements. Images show that the protein immediately started to accumulate rapidly on the membranes, likely due to favorable hydrophobic interactions between the PVDF and protein, followed by decreasing rates of fouling with time as protein accumulated on the membrane surface. The knowledge generated in this study can be used to design membranes that inhibit fouling or otherwise direct foulants to deposit selectively in regions that minimize loss of flux.

Understanding the Role of Pattern Geometry on Nanofiltration Threshold Flux.

Colloidal fouling can be mitigated by membrane surface patterning. This contribution identifies the effect of different pattern geometries on fouling behavior. Nanoscale line-and-groove patterns with different feature sizes were applied by thermal embossing on commercial nanofiltration membranes. Threshold flux values of as-received, pressed, and patterned membranes were determined using constant flux, cross-flow filtration experiments. A previously derived combined intermediate pore blocking and cake filtration model was applied to the experimental data to determine threshold flux values. The threshold fluxes of all patterned membranes were higher than the as-received and pressed membranes. The pattern fraction ratio (PFR), defined as the quotient of line width and groove width, was used to analyze the relationship between threshold flux and pattern geometry quantitatively. Experimental work combined with computational fluid dynamics simulations showed that increasing the PFR leads to higher threshold flux. As the PFR increases, the percentage of vortex-forming area within the pattern grooves increases, and vortex-induced shielding increases. This study suggests that the PFR should be higher than 1 to produce patterned membranes with maximal threshold flux values. Knowledge generated in this study can be applied to other feature types to design patterned membranes for improved control over colloidal fouling.