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.