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Living systems are characterized by their spatially highly inhomogeneous nature which is susceptible to modify fundamentally the behavior of biomolecular species, including the proteins that underpin biological functionality in cells. Spatial gradients in chemical potential are known to lead to strong transport effects for colloidal particles, but their effect on molecular scale species such as proteins has remained largely unexplored. Here, we improve on existing diffusiophoresis microfluidic technique to measure protein diffusiophoresis in real space. The measurement of proteins is made possible by two ameliorations. First, a label-free microscope is used to suppress label interference. Second, improvements in numerical methods are developed to meet the particular challenges posed by small molecules. We demonstrate that individual proteins can undergo strong diffusiophoretic motion in salt gradients in a manner which is sufficient to overcome diffusion and which leads to dramatic changes in their spatial organization on the scale of a cell. Moreover, we demonstrate that this phenomenon can be used to control the motion of proteins in microfluidic devices. These results open up a path towards a physical understanding of the role of gradients in living systems in the spatial organization of macromolecules and highlight novel routes towards protein sorting applications on device.

Funder: Herchel Smith Fund, Funder: St John’s College Cambridge, Funder: Centre for Misfolding Diseases, Cambridge, Funder: Swiss FCS and the Forschungskredit of the University of Zurich, Funder: Frances and Augustus Newman Foundation, Funder: BBRSC, Funder: NOMIS Foundation, The clinical outcome of SARS-CoV-2 infections, which can range from asymptomatic to lethal, is crucially shaped by the concentration of antiviral antibodies and by their affinity to their targets. However, the affinity of polyclonal antibody responses in plasma is difficult to measure. Here we used microfluidic antibody affinity profiling (MAAP) to determine the aggregate affinities and concentrations of anti-SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 19 of which were healthy donors, 20 displayed mild symptoms, and 3 were critically ill. We found that dissociation constants, K d, of anti-receptor-binding domain antibodies spanned 2.5 orders of magnitude from sub-nanomolar to 43 nM. Using MAAP we found that antibodies of seropositive individuals induced the dissociation of pre-formed spike-ACE2 receptor complexes, which indicates that MAAP can be adapted as a complementary receptor competition assay. By comparison with cytopathic effect-based neutralisation assays, we show that MAAP can reliably predict the cellular neutralisation ability of sera, which may be an important consideration when selecting the most effective samples for therapeutic plasmapheresis and tracking the success of vaccinations.

3D scaffolds in the form of hydrogels and microgels have allowed for more native cell-culture systems to be developed relative to flat substrates. Native biological tissues are, however, usually spatially inhomogeneous and anisotropic, but regulating the spatial density of hydrogels at the microscale to mimic this inhomogeneity has been challenging to achieve. Moreover, the development of biocompatible synthesis approaches for protein-based microgels remains challenging, and typical gelation conditions include UV light, extreme pH, extreme temperature, or organic solvents, factors which can compromise the viability of cells. This study addresses these challenges by demonstrating an approach to fabricate protein microgels with controllable radial density through microfluidic mixing and physical and enzymatic crosslinking of gelatin precursor molecules. Microgels with a higher density in their cores and microgels with a higher density in their shells are demonstrated. The microgels have robust stability at 37 °C and different dissolution rates through enzymolysis, which can be further used for gradient scaffolds for 3D cell culture, enabling controlled degradability, and the release of biomolecules. The design principles of the microgels could also be exploited to generate other soft materials for applications ranging from novel protein-only micro reactors to soft robots.

Many biological molecules are by their nature amphiphilic and have the ability to act as surfactants, stabilizing interfaces between aqueous and immiscible oil phases. In this paper, we explore the adsorption kinetics of surfactin, a naturally occurring cyclic lipopeptide, at hexadecane/water interfaces and compare and contrast its adsorption behaviour with that of synthetic alkyl benzene sulfonate isomers, through direct measurements of changes in interfacial tension upon surfactant adsorption. We access millisecond time resolution in kinetic measurements by making use of droplet microfluidics to probe the interfacial tension of hexadecane droplets dispersed in a continuous water phase through monitoring their deformation when the droplets are exposed to shear flows in a microfluidic channel with regular corrugations. Our results reveal that surfactin rapidly adsorbs to the interface, thus the interfacial tension equilibrates within 300 ms, while the synthetic surfactants used undergo adsorption processes at an approximately one order of magnitude longer timescale. The approach presented may provide opportunities for understanding and modulating the adsorption mechanism of amphiphiles on a variety of interfaces in the context of life sciences and industrial applications.