Performance of various 3D-printed monolith geometries as an alternative to expanded bed adsorption for protein purification.

Here, we compare the performance of various three-dimensional-printed Monolith Adsorption (PMA) columns designed from a triply periodic minimal surface geometry, the Schoen gyroid. The structures examined had designed hydraulic diameters between 203 and 458 µm and voidages of 40%-60%. We compare column efficiency, porosity, static binding capacity and dynamic binding capacity for various load volumes and flow rates. The results show that all structures allowed efficient passage of yeast cells (>97%) over a wide range of interstitial velocities (191 to 1911 cm/h) while maintaining a low pressure drop (<0.1 MPa). The structure with a voidage of 40% and a hydraulic diameter of 203 µm showed the best performance in all aspects evaluated. Bovine serum albumin (BSA) recoveries for all structures (27%-91% when the loaded volume was 180 mL) were significantly affected by hydraulic diameter, mean channel wall thickness, velocity and voidage. Moreover, biomass addition resulted in a decrease in BSA recovery, which became more obvious at high velocities. However, this did not lead to a dramatic reduction in saturated binding capacity, significant changes in axial dispersion, or blockage of channels and could be compensated for by recirculation of the feed, even at high velocity. PMA thus potentially provides an appealing alternative to Expanded Bed Adsorption, retaining the latter's advantages, while eliminating fluidization issues and minimizing both processing time and buffer consumption.

A numerical investigation of the hydrodynamic dispersion in triply periodic chromatographic stationary phases.

3D printed custom chromatographic stationary phases have recently been demonstrated. Using the Lattice Boltzmann Method, we compared the model-predicted chromatographic performance of random packing of monodisperse spheres, open tubular columns (OTC) and stationary phases based on three triply periodic minimal surfaces (TPMS): Schwarz Diamond (SD), Schoen Gyroid (SG) and Schwarz Primitive (SP). Three performance metrics were employed in this comparison: i) reduced plate height, ii) Darcy number, iii) kinetic performance factor. Each simulated geometry was unconfined with an impermeable stationary phase to remove wall effects and pore diffusion. The performance was studied for macro-porosities in the range 0.2 to 0.8, depending on the geometry. OTCs were found to have superior permeability to both random sphere packing and TPMS structures across the entire porosity range. At porosity greater than 0.366, the Schwarz Diamond medium achieved the lowest levels of band broadening and greatest kinetic performance. The reduced plate height of all stationary phase geometries was shown to increase with bed porosity. The kinetic performance was found to increase with porosity for TPMS structures, decrease with porosity for random packing and be independent of porosity for OTCs. This work illustrates that chromatographic stationary phase geometries based on TPMS structures are theoretically competitive with random packing and open tubular columns and their feasibility for practical chromatography should continue to be explored.

Use of a Novel DNA-Loaded Alginate-Calcium Carbonate Biopolymer Surrogate to Study the Engulfment of Legionella pneumophila by Acanthamoeba polyphaga in Water Systems.

The engulfment of Legionella pneumophila by free-living amoebae (FLA) in engineered water systems (EWS) enhances L. pneumophila persistence and provides a vehicle for rapid replication and increased public health risk. Despite numerous legionellosis outbreaks worldwide, effective tools for studying interactions between L. pneumophila and FLA in EWS are lacking. To address this, we have developed a biopolymer surrogate with a similar size, shape, surface charge, and hydrophobicity to those of stationary-phase L. pneumophila. Parallel experiments were conducted to observe the engulfment of L. pneumophila and the surrogate by Acanthamoeba polyphaga in dechlorinated, filter-sterilised tap water at 30°C for 72 h. Trophozoites engulfed both the surrogate and L. pneumophila, reaching maximum uptake after 2 and 6 h, respectively, but the peak surrogate uptake was ~2-log lower. Expulsion of the engulfed surrogate from A. polyphaga was also faster compared to that of L. pneumophila. Confocal laser scanning microscopy confirmed that the surrogate was actively engulfed and maintained within vacuoles for several hours before being expelled. L. pneumophila and surrogate phagocytosis appear to follow similar pathways, suggesting that the surrogate can be developed as a useful tool for studying interactions between L. pneumophila and FLA in EWS. IMPORTANCE The internalization of L. pneumophila within amoebae is a critical component of their life cycle in EWS, as it protects the bacteria from commonly used water disinfectants and provides a niche for their replication. Intracellularly replicated forms of L. pneumophila are also more virulent and resistant to sanitizers. Most importantly, the bacteria's adaptation to the intracellular environments of amoebae primes them for the infection of human macrophages, posing a significant public health risk in EWS. The significance of our study is that a newly developed L. pneumophila biopolymer surrogate can mimic the L. pneumophila engulfment process in A. polyphaga, a free-living amoeba. With further development, the surrogate has the potential to improve the understanding of amoeba-mediated L. pneumophila persistence in EWS and the associated public health risk management.

Evaluation of Biopolymer Materials and Synthesis Techniques to Develop a Rod-Shaped Biopolymer Surrogate for Legionella pneumophila.

Biopolymer microparticles have been developed for applications that require biocompatibility and biodegradability, such as drug delivery. In this study, we assessed the production of microparticles using carnauba wax, κ-carrageenan, alginate, and poly (lactic-co-glycolic acid) (PLGA) with the aim of developing a novel, DNA-tracer-loaded, biopolymer surrogate with a size, shape, surface charge, and relative hydrophobicity similar to stationary-phase Legionella pneumophila to mimic the bacteria's mobility and persistence in engineered water systems. We found that the type and concentration of biopolymer, reaction conditions, and synthesis methods affected the morphology, surface charge, relative hydrophobicity, and DNA tracer loading efficiency of the biopolymer microparticles produced. Carnauba wax, κ-carrageenan, and alginate (Protanal®, and low and medium viscosity) produced highly polydisperse microspheres. In contrast, PLGA and alginate-CaCO3 produced uniform microspheres and rod-shaped microparticles, respectively, with high DNA tracer loading efficiencies (PLGA 70% and alginate-CaCO3 95.2 ± 5.7%) and high reproducibilities. Their synthesis reproducibility was relatively high. The relative hydrophobicity of PLGA microspheres closely matched the cell surface hydrophobicity of L. pneumophila but not the bacterial morphology, whereas the polyelectrolyte layer-by-layer assembly was required to enhance the relative hydrophobicity of alginate-CaCO3 microparticles. Following this surface modification, alginate-CaCO3 microparticles represented the best match to L. pneumophila in size, morphology, surface charge, and relative hydrophobicity. This new biopolymer surrogate has the potential to be used as a mimic to study the mobility and persistence of L. pneumophila in water systems where the use of the pathogen is impractical and unsafe.

Capillaric field effect transistors.

Controlling fluid flow in capillaric circuits is a key requirement to increase their uptake for assay applications. Capillary action off-valves provide such functionality by pushing an occluding bubble into the channel using a difference in capillary pressure. Previously, we utilized the binary switching mode of this structure to develop a powerful set of fundamental fluidic valving operations. In this work, we study the transistor-like qualities of the off-valve and provide evidence that these structures are in fact functionally complementary to electronic junction field effect transistors. In view of this, we propose the new term capillaric field effect transistor to describe these types of valves. To support this conclusion, we present a theoretical description, experimental characterization, and practical application of analog flow resistance control. In addition, we demonstrate that the valves can also be reopened. We show modulation of the flow resistance from fully open to pinch-off, determine the flow rate-trigger channel volume relationship and demonstrate that the latter can be modeled using Shockley's equation for electronic transistors. Finally, we provide a first example of how the valves can be opened and closed repeatedly.

Molecular basis of a redox switch: molecular dynamics simulations and surface plasmon resonance provide insight into reduced and oxidised angiotensinogen.

Angiotensinogen fine-tunes the tightly controlled activity of the renin-angiotensin system by modulating the release of angiotensin peptides that control blood pressure. One mechanism by which this modulation is achieved is via angiotensinogen's Cys18-Cys138 disulfide bond that acts as a redox switch. Molecular dynamics simulations of each redox state of angiotensinogen reveal subtle dynamic differences between the reduced and oxidised forms, particularly at the N-terminus. Surface plasmon resonance data demonstrate that the two redox forms of angiotensinogen display different binding kinetics to an immobilised anti-angiotensinogen monoclonal antibody. Mass spectrometry mapped the epitope for the antibody to the N-terminal region of angiotensinogen. We therefore provide evidence that the different redox forms of angiotensinogen can be detected by an antibody-based detection method.

Printed monolith adsorption as an alternative to expanded bed adsorption for purifying M13 bacteriophage.

An ordered 3D printed chromatography stationary phase was used to purify M13 bacteriophage (M13) directly from crude cell culture. This new approach, which offers the same advantages as expanded bed adsorption (EBA) with regard to tolerating solids-laden feed streams but without the corresponding issues associated with fluidized bed stability that affect the latter, can be described as "printed monolith adsorption (PMA)". PMA columns (5, 10 and 15 cm length by 1 cm diameter) were made via a wax templating method from cross-linked cellulose hydrogel and functionalized with a quaternary amine ligand. The recovery of M13 was found to be strongly linked to load flow rate, with the highest recovery 89.7% ± 6% for 1.4 × 1011 pfu/mL of resin occurring at 76 cm/h with a 10 cm column length. A recovery of 87.7% ± 5% for 1.49 × 1011 pfu/mL of media was achieved with a 15 cm column length under conditions comparable to a reported EBA process. The PMA process was completed three times faster than EBA because PMA flow rates can readily be adjusted during operation, with high flow rates and low back pressure, which is unique to the ordered monolithic media geometry used. Equilibration, wash, and cleaning steps were carried out at high flow rates (611 cm/h), minimizing process time and were limited only by the volumetric flow rate capacity of the pumps used, rather than column back pressure (<0.1 MPa at 611 cm/hr). Initial capture of M13 appears to occur on the surface of the monolith solid phase (i.e. the mobile phase channel walls) and subsequently, at a slower rate, within the internal pores of the solid phase media. The difference in binding rate between these two sites is likely caused by slow pore diffusion of the large M13 particles into the pores, with similar slow diffusion out of the pores resulting in tailing of the elution peak. The results indicate that PMA is a promising technology for the efficient purification of viruses directly from crude cell culture.

New flow control systems in capillarics: off valves.

Capillary systems are a promising technology for point-of-care microfluidics, since they are pre-programmable and self-powered. This work introduces "off valves" as a key building block for capillaric circuits, providing easy-to-use, multi-purpose valving functionality and autonomous flow control. To this end we present a set of switching valve designs that use trigger channels and liquid input alone to close or open connections between channels in a highly controllable fashion. The key element of all these valve designs is a new off trigger valve, which is characterised in detail here and holds the potential for transistor-like switching and resistance tuning. As an example for the potential applications of switching valves, we demonstrate how they can be used for flow resistance control in a complex microfluidic circuit and for sequential chemical loading into a reaction chamber. Use of the switching valves for the latter in particular allowed for the tuning of incubation times and volumetric measurement, thus confirming applicability of the valves for automated and self-powered immunoassays in point-of-care environments.

Complex Geometry Cellulose Hydrogels Using a Direct Casting Method.

To facilitate functional hydrogel part production using the indirect wax mould method, it is necessary to understand the relationships between materials, process and mould removal. This research investigated the thermophysical properties, wettability and surface roughness of wax template moulds in the production of cellulose hydrogel objects. Cellulose gel was thermally formed and shaped in three different wax moulds-high melting point paraffin, sacrificial investment casting wax and Solidscape® wax-by physical cross-linking of polymer networks of cellulose solution in NaOH/urea aqueous solvent. All three wax moulds were capable of casting cellulose hydrogel objects. Cellulose gelling time was reduced by increasing the temperature. Thus, the mould melting temperature had a direct effect on the gelling time. It was found that mould removal time varied based on the contact angle (CA) of the cellulose solution and the mould, and based on the melting point of the mould. A higher CA of cellulose solution on the wax moulds resulted in faster mould removal. When melting the wax in 90 °C water, high melting point paraffin, sacrificial investment casting and Solidscape® wax took about 3, 2 and 1½ h, respectively, to remove the moulds from the cellulose gel.

3D Printing of Gelled and Cross-Linked Cellulose Solutions, an Exploration of Printing Parameters and Gel Behaviour.

In recent years, 3D printing has enabled the fabrication of complex designs, with low-cost customization and an ever-increasing range of materials. Yet, these abilities have also created an enormous challenge in optimizing a large number of process parameters, especially in the 3D printing of swellable, non-toxic, biocompatible and biodegradable materials, so-called bio-ink materials. In this work, a cellulose gel, made out of aqueous solutions of cellulose, sodium hydroxide and urea, was used to demonstrate the formation of a shear thinning bio-ink material necessary for an extrusion-based 3D printing. After analysing the shear thinning behaviour of the cellulose gel by rheometry a Design of Experiments (DoE) was applied to optimize the 3D bioprinter settings for printing the cellulose gel. The optimum print settings were then used to print a human ear shape, without a need for support material. The results clearly indicate that the found settings allow the printing of more complex parts with high-fidelity. This confirms the capability of the applied method to 3D print a newly developed bio-ink material.

Numerical Elucidation of Flow and Dispersion in Ordered Packed Beds: Nonspherical Polygons and the Effect of Particle Overlap on Chromatographic Performance.

Spherical particles are widely considered as the benchmark stationary phase for preparative and analytical chromatography. Although this has proven true for randomly packed beds in the past, we challenge this paradigm for ordered packings, the fabrication of which are now feasible through additive manufacturing (3D printing). Using computational fluid dynamics (Lattice Boltzmann Method) this work shows that nonspherical particles can both reduce mobile-phase band broadening and increase permeability compared with spheres in ordered packed beds. In practice, ordered packed beds can only remain physically stable if the particles are fused to form a contiguous matrix, thus creating a positional overlap at the points of fusion between what would otherwise be discrete particles. Overlap is shown to decrease performance of ordered packed beds in all observed cases, thus we recommend it should be kept to the minimum extent necessary to ensure physical stability. Finally, we introduce a metric to estimate column performance, the mean deviated velocity, a quantitative description of the spread of the velocity field in the column. This metric appears to be a good indicator of mobile-phase dispersion in ordered packed bed media, including overlapped beds, and is a useful tool for screening new stationary-phase morphologies without having to perform computationally expensive simulations.

Analysis of the Effect of Processing Conditions on Physical Properties of Thermally Set Cellulose Hydrogels.

Cellulose-based hydrogels were prepared by dissolving cellulose in aqueous sodium hydroxide (NaOH)/urea solutions and casting it into complex shapes by the use of sacrificial templates followed by thermal gelation of the solution. Both the gelling temperatures used (40⁻80 °C), as well as the method of heating by either induction in the form of a water bath and hot press or radiation by microwaves could be shown to have a significant effect on the compressive strength and modulus of the prepared hydrogels. Lower gelling temperatures and shorter heating times were found to result in stronger and stiffer gels. Both the effect of physical cross-linking via the introduction of additional non-dissolving cellulosic material, as well as chemical cross-linking by the introduction of epichlorohydrin (ECH), and a combination of both applied during the gelation process could be shown to affect both the mechanical properties and microstructure of the hydrogels. The added cellulose acts as a physical-cross-linking agent strengthening the hydrogen-bond network as well as a reinforcing phase improving the mechanical properties. However, chemical cross-linking of an unreinforced gel leads to unfavourable bonding and cellulose network formation, resulting in drastically increased pore sizes and reduced mechanical properties. In both cases, chemical cross-linking leads to larger internal pores.

Modelling ordered packed beds of spheres: The importance of bed orientation and the influence of tortuosity on dispersion.

Ordered packing has previously been considered for porous media applications in the industrial and analytical worlds, with implementation constrained only by the lack of feasible fabrication methods. Additive manufacturing now provides the answer to this limitation, which leads to the novel domain of customized ordered packing and a variety of optimized geometries. In this work, the chromatographic behaviour of ordered configurations of particles was described using computational fluid dynamics methods based on the Lattice Boltzmann Model. The model was first validated by matching van Deemter trends for ordered and random packings shown in previous research. The influence of rotations of the ordered configurations was then considered, indicating that orientational changes with respect to the main flow axis can strongly affect minimum plate height. In particular, it is demonstrated that targeted rotations of ordered packings can reduce axial dispersion while improving transverse dispersion, thus improving chromatographic performance. This principle is clearly shown in a strong linear correlation between tortuosity and plate height, offering an additional parameter to enable a priori control of the performance of ordered packings. Furthermore, rotation of the packing does not change porosity or surface area and has a relatively small effect on permeability. Thus, highly permeable packings with poor dispersion can be improved in terms of chromatographic impedance by simple rotation of the packing orientation. This work further demonstrates the advantages of ordered packings over randomly packed beds, and introduces new perspectives on the development of chromatographic structures with improved performance.

DNA G-quadruplexes show strong interaction with DNA methyltransferases in vitro.

The DNA methyltransferase enzymes (DNMTs) catalyzing cytosine methylation do so at specific locations of the genome, although with some level of redundancy. The de novo methyltransferases DNMT3A and 3B play a vital role in methylating the genome of the developing embryo in regions devoid of methylation marks. The ability of DNMTs to colocalize at sites of DNA damage is suggestive that recognition of mispaired bases and unusual structures is inherent to the function of these proteins. We provide evidence for G-quadruplex formation within imprinted gene promoters, and report high-affinity binding of recombinant human DNMTs to such DNA G-quadruplexes in vitro. These observations suggest a potential interaction of G-quadruplexes with the DNA methylation machinery, which may be of epigenetic and biological significance.

Corticosteroid-binding globulin (CBG) reactive centre loop antibodies and surface plasmon resonance interrogate the proposed heat dependent "flip-flop" mechanism of human CBG.

Corticosteroid-binding globulin (CBG) is the predominant carrier of cortisol in circulation and is a non-inhibitory member of the serpin superfamily of serine protease inhibitors. In the stressed or "S" conformation, CBG possesses an intact exposed reactive centre loop (RCL) that can be irreversibly cleaved by elastase released from activated human neutrophils whereupon it adopts a relaxed or "R" conformation. The latter conformation has decreased affinity for cortisol, allowing the release of the majority of cortisol at sites of inflammation. Recently there has been speculation that mild increments in heat such as found in pyrexia (39-40°C) may also induce a reversible "flip-flop" of the RCL into the body of the protein structure, without cleavage, facilitating a reversible temperature-dependent release of cortisol. Here we raised a new monoclonal antibody to the RCL of human CBG and used this in concert with an existing RCL antibody and show by surface plasma resonance that, at temperatures up to 40°C, the RCL of purified CBG and the RCL of CBG in intact plasma is accessible to these two antibodies. Together, the epitopes of these antibodies span 11 consecutive amino acids (STGVTLNLTSK) of the 18 residues of the RCL. This adequate antibody cover of the RCL sequence leads to the conclusion that the proposed temperature-dependent "flip-flop" of the RCL of CBG is doubtful.

Adsorption of chemically synthesized mussel adhesive peptide sequences containing DOPA on stainless steel.

The adsorption of proteins at solid-liquid interfaces is important in biosensor and biomaterial applications. Marine mussels affix themselves to surfaces using a highly cross-linked, protein-based adhesive containing a high proportion of L-3,4-dihydroxyphenylalanine (DOPA) residues. In this work, the effect of DOPA residues on protein adhesion on stainless steel surfaces was studied using a quartz crystal microbalance with dissipation system. The adsorption of two repetitive peptide motifs, KGYKYYGGSS and KGYKYY, from the mussel Mytilus edulis foot protein 5 on stainless steel was studied before and after chemo-enzymatic modification of tyrosine residues to DOPA using mushroom tyrosinase. Conversion from tyrosine to DOPA, evaluated by HPLC, was in the range 70-99%. DOPA-modified sequences showed fourfold greater adhesion than unmodified M. edulis foot protein 5 motifs.

A surface plasmon resonance assay for measurement of neuraminidase inhibition, sensitivity of wild-type influenza neuraminidase and its H274Y mutant to the antiviral drugs zanamivir and oseltamivir.

Antiviral resistance is currently monitored by a labelled enzymatic assay, which can give inconsistent results because of the short half-life of the labelled product, and variations in assay conditions. In this paper, we describe a competitive surface plasmon resonance (SPR) inhibition assay for measuring the sensitivities of wild-type neuraminidase (WT NA) and the H274Y (histidine 274 tyrosine) NA mutant to antiviral drugs. The two NA isoforms were expressed in High-five™ (Trichoplusia ni) insect cells. A spacer molecule (1,6-hexanediamine (HDA)) was conjugated to the 7-hydroxyl group of zanamivir, and the construct (HDA-zanamivir) was immobilized onto a SPR sensor chip to obtain a final immobilization response of 431 response units. The immobilized HDA-zanamivir comprised a bio-specific ligand for the WT and mutant proteins. The effects of the natural substrate (sialic acid) and two inhibitors (zanamivir and oseltamivir) on NA binding to the immobilized ligand were studied. The processed SPR data was analysed to determine 50% inhibitory concentrations (IC50-spr ), using a log dose-response curve fit. Although both NA isoforms had almost identical IC50-spr values for sialic acid (WT = 5.5 nM; H274Y mutant = 3.25 nM) and zanamivir (WT = 2.16 nM; H274Y mutant = 2.42 nM), there were significant differences between the IC50-spr values obtained for the WT (7.7 nM) and H274Y mutant (256 nM) NA in the presence of oseltamivir, indicating that oseltamivir has a reduced affinity for the H274Y mutant. The SPR inhibition assay strategy presented in this work could be applied for the rapid screening of newly emerging variants of NA for their sensitivity to antiviral drugs.

Development of a surface plasmon resonance assay to measure the binding affinity of wild-type influenza neuraminidase and its H274Y mutant to the antiviral drug zanamivir.

Influenza is one of the most common infections of the upper respiratory tract. Antiviral drugs that are currently used to treat influenza, such as oseltamivir and zanamivir, are neuraminidase (NA) inhibitors. However, the virus may develop resistance through single-point mutations of NA. Antiviral resistance is currently monitored by a labelled enzymatic assay, which can be inconsistent because of the short half-life of the labelled product and variations in the assay conditions. In this paper, we describe a label-free surface plasmon resonance (SPR) assay for measuring the binding affinity of NA-drug interactions. Wild-type (WT) NA and a histidine 274 tyrosine (H274Y) mutant were expressed in High Five™ (Trichoplusia ni) insect cells. A spacer molecule (1,6-hexanediamine) was site-specifically conjugated to the 7-hydroxyl group of zanamivir, which is not involved in binding to NA, and the construct was immobilized onto a SPR sensor Chip to obtain a final immobilization response of 431 response units. Binding responses obtained for WT and H274Y mutant NAs were fitted to a simple Langmuir 1:1 model with drift to obtain the association (ka ) and dissociation (kd ) rate constants. The ratio between the binding affinities for the two isoforms was comparable to literature values obtained using labelled enzyme assays. Significant potential exists for an extension of this approach to test for drug resistance of further NA mutants against zanamivir and other antiviral drugs, perhaps paving the way for a reliable SPR biosensor assay that may replace labelled enzymatic assays.

Reversible and rapid pH-regulated self-assembly of a poly(ethylene glycol)-peptide bioconjugate.

The use of external triggers to manipulate the secondary structure of self-assembling peptides conjugated to flexible synthetic polymers is a challenging problem, particularly in terms of reversibility. Here, we demonstrate, for the first time, sustained rapid and reversible, pH-regulated self-assembly of the peptide ELELELELELF (EL-5F) and its conjugates with 2 and 5 kDa poly(ethylene glycol) (EL-5F-PEG-2K and EL-5F-PEG-5K). Circular dichroism indicated the formation of ß-sheet structures at pH < 5.9, 5.8, and 5.4 and disassembly to random coils above those pH values for EL-5F, EL-5F-PEG-2K, and EL-5F-PEG-5K, respectively. ß-sheets were confirmed by the thioflavin T assay, while transmission electron microscopy revealed the existence of extended fibrillar structures below the above pH values. pH-induced secondary structure conversion was reproducible for over 15 cycles, even at salt concentrations of up to 200 mM NaCl, and was quantitatively related to the pH. Self-supporting hydrogelation after self-assembly was observed at concentrations as low as 0.2 wt %, which is 15-fold lower than previously reported concentrations. This simple approach to mediate reversible self-assembly of EL-5F-PEG bioconjugates is expected to offer novel functionality relevant to drug delivery and bioseparation systems.