Figure 8. (a) Accelerated processing times for four enzymes as indicated, (b) reaction landscape for simultaneous changes in enzyme and substrate concentrations, (c) increments of 50 rpm for scans of rotational speeds, (d) alkaline phosphatase and substrate being processed with the tilt angle varied, (e) catalysis of alkaline phosphate affected by the addition of PEG, (f) lysozyme activity for boiled egg white after processing by the VFD per mg protein, with higher lysozyme, recovered activities represented by larger circles and lower activity represented by smaller circles, (g) CD spectra of denatured HEWL before and after being refolded by the VFD (h) solution of protein introduced at the bottom of the tube, and collection of folded proteins at the top of the VFD in continuous mode, (i) lysozyme activity of native and recombinantly expressed HEWL after refolding by the VFD per mg protein. (a-e) Reproduced with permission24. Copyright 2016, John Wiley and Sons (f-i) Reproduced with permission27. Copyright 2015, John Wiley and Sons.
Besides protein refolding, the VFD provides a novel processing methodology for phase demixing for the separation of proteins, as established for an aqueous two-phase system (ATPS) of aqueous potassium phosphate and polyethylene glycol, for a mixture of C-phycocyanin (C-PC)Spirulina maxima 28. The process increased the efficacy of separation by 22% compared to conventional ATPS methods, with a 1.18-fold increase in C-PC purity in contrast to allophycocyanin, the primary contaminant protein, and a 9.3-fold increase in phase demixing efficiency, Figure 9. This VFD based methodology has potential for rapid phase demixing associated with purifying biologically active proteins. A mechanistic understanding of phase separation in VFD was established in 2022.14 This is driven by micron to submicron size topological flow regimes in the thin films in VFD which induces high inter-phase mass transfer and facilitates the protein exchange. Briefly, this is achieved through a hemispherical base tube which creates a Coriolis force as a spinning top (ST) fluid flow in the less dense liquid which penetrates the denser layer of liquid, delivering liquid from the upper layer through the lower layer to the surface of the tube. In the same way, Faraday wave induced double helical flows penetrate both phases, also facilitating mass transfer across the phase boundary. Therefore, protein separation can be achieved instantaneously as a mixture with no evidence of damage to the proteins under the shearing.
Furthermore, Britton et al. reported utilizing thin film processing in a continuous mode as a versatile strategy for immobilizing enzymes, with glutaraldehyde and non-covalent bioconjugation immobilizing enzymes onto the surface of the tubes52. The stock protein solution was able to consecutively coat more than ten reactors even after being recycled, with the approach requiring only a nanogram of protein for each tube. This technique reduced the number of necessary proteins and a piranha-cleaning solution and other reagents by up to 96% by confining the reagents in thin films during immobilization, with no loss in catalytic activity after being processed for ten hours, Figure 9. Nature’s machinery can generate complex molecules by incorporating biocatalysts into multistep processes, offering the potential for proteins with low expression to facilitate biocatalysts for complex substrate transformations in pharmaceutical manufacture. Another example of lysing/disrupting cells using VFD is for the biodiesel production.53,54 The performance of the processing technology, both in confined and continuous flow mode, was evaluated by assessing the efficiencies of fatty acid extraction and the subsequent conversion of fatty acids to fatty acid methyl ester (FAME). Average extraction efficacies were 41% and conversion efficiencies >90% with the processing technology showing a broad tolerance to parameter settings.
The VFD is also capable of probing the structure of self-organized systems55. As reported by Mo et al ., vesicles 107 ± 19 nm in diameter, shown in Figure 9, are found on the self-assembly of phenolic oxygen centers with tetra-para-phosphonomethyl calix[4]-arene bearing n-hexyl moiety attachments, were useful within a macrocycle cavity by binding to carboplatin under the shear stress induced by the dynamic thin films within the VFD. Carboplatin was retained inside the calixarene’s hierarchical cavity structure, and the diameters of the vesicles were maintained after shearing. The loading efficiency and release profile of the carboplatin were investigated, with the drugs loaded under shear. The vesicles are stable at a normal tissue pH with preferential release of the drug cargo at pH 6, which is the typical pH for cancerous tissues. Thus they have the potential for anticancer drug delivery applications. The hierarchical structured vesicles lower the concentration of IC50 10-fold, increasing carboplatin efficacy 4.5-fold for ovarian cancer cells and increasing cell percentage during DNA replication (S-phase) of the cell cycle. The vesicles mimic calixarene lipids and have potential application in drug delivery. This would only increase once a targeting agent was tagged alongside, such as fluorescent molecules with aggregation-induced emission characteristics.