A programmable and automated optical electrowetting-on-dielectric (oEWOD) driven platform for massively parallel and sequential processing of single cell assay operations.

Recently, there has been an increasing emphasis on single cell profiling for high-throughput screening workflows in drug discovery and life sciences research. However, the biology underpinning these screens is often complex and is insufficiently addressed by singleplex assay screens. Traditional single cell screening technologies have created powerful sets of 'omic data that allow users to bioinformatically infer biological function, but have as of yet not empowered direct functional analysis at the level of each individual cell. Consequently, screening campaigns often require multiple secondary screens leading to laborious, time-consuming and expensive workflows in which attrition points may not be queried until late in the process. We describe a platform that harnesses droplet microfluidics and optical electrowetting-on-dielectric (oEWOD) to perform highly-controlled sequential and multiplexed single cell assays in massively parallelised workflows to enable complex cell profiling during screening. Soluble reagents or objects, such as cells or assay beads, are encapsulated into droplets of media in fluorous oil and are actively filtered based on size and optical features ensuring only desirable droplets (e.g. single cell droplets) are retained for analysis, thereby overcoming the Poisson probability distribution. Droplets are stored in an array on a temperature-controlled chip and the history of individual droplets is logged from the point of filter until completion of the workflow. On chip, droplets are subject to an automated and flexible suite of operations including the merging of sample droplets and the fluorescent acquisition of assay readouts to enable complex sequential assay workflows. To demonstrate the broad utility of the platform, we present examples of single-cell functional workflows for various applications such as antibody discovery, infectious disease, and cell and gene therapy.

Single-Molecule Stoichiometry of Supramolecular Complexes.

The use of single-molecule microscopy is introduced as a method to quantify the photophysical properties of supramolecular complexes rapidly at ultra low concentrations (<1 nM), previously inaccessible. Using a model supramolecular system based on the host-guest complexation of cucurbit[n]uril (CB[n]) macrocycles together with a fluorescent guest (Ant910Me), we probe fluorescent CB[n] host-guest complexes in the single molecule regime. We show quantification and differentiation of host-guest photophysics and stoichiometries, both in aqueous media and noninvasively in hydrogel, by thresholding detected photons. This methodology has wide reaching implications in aiding the design of next-generation materials with programmed and controlled properties.

Textured Microcapsules through Crystallization.

This work demonstrates the fabrication of surface-textured microcapsules formed from emulsion droplets, which are stabilized by an interlocking mesh of needle-like crystals. Crystals of the small-organic-compound decane-1,10-bis(cyclohexyl carbamate) are formed within the geometric confinement of the droplets, through precipitation from a binary-solvent-dispersed phase. This binary mixture consists of a volatile solvent and nonvolatile carrier oil. Crystallization is facilitated upon supersaturation due to evaporation of the volatile solvent. Microcapsule diameter can be easily tuned using microfluidics. This approach also proves to be scalable when using conventional mixers, yielding spikey microcapsules with diameters in the range of 10-50 µm. It is highlighted that the capsule shape can be molded and arrested by jamming using recrystallization in geometric confinement. Moreover, it is shown that these textured microcapsules show a promising enhanced deposition onto a range of fabric fibers.

Synthesis of Janus and Patchy Particles Using Nanogels as Stabilizers in Emulsion Polymerization.

Polymer nanogels are used as colloidal stabilizers in emulsion polymerization. The nanogels were made by the covalent cross-linking of block copolymer micelles, the macromolecular building blocks of which were synthesized using a combination of catalytic chain transfer emulsion polymerization and reversible addition-fragmentation chain transfer (RAFT) of methacrylate monomers. The use of the nanogels in an emulsion polymerization led to anisotropic Janus and patchy colloids, where a latex particle was decorated by a number of patches on its surface. Control of the particle size and patch density was achieved by tailoring of the reaction conditions, such as varying the amount of nanogels, pH, and salt concentration. Overall, the emulsion polymerization process in the presence of nanogels as stabilizers is shown to be a versatile and easily scalable route toward the fabrication of Janus and patchy polymer colloids.

Improving the engine power of a catalytic Janus-sphere micromotor by roughening its surface.

Microspheres with catalytic caps have become a popular model system for studying self-propelled colloids. Existing experimental studies involve predominantly "smooth" particle surfaces. In this study we determine the effect of irregular surface deformations on the propulsive mechanism with a particular focus on speed. The particle surfaces of polymer microspheres were deformed prior to depositing a layer of platinum which resulted in the formation of nanoscopic pillars of catalyst. Self-propulsion was induced upon exposure of the micromotors to hydrogen peroxide, whilst they were dispersed in water. The topological surface features were shown to boost speed (~2×) when the underlying deformations are small (nanoscale), whilst large deformations afforded little difference despite a substantial apparent catalytic surface area. Colloids with deformed surfaces were more likely to display a mixture of rotational and translational propulsion than their "smooth" counterparts.

Roughening up polymer microspheres and their diffusion in a liquid.

A simple, versatile approach for the roughening of polymer microparticles surfaces via a deformation technique in the presence of an inorganic matrix is presented here. The process consists of straightforward steps: (1) preparation of a bicomposite colloidal sol, that is polymer particles and inorganic particles, dispersed in a liquid, (2) drying of the mixture onto a suitable hard substrate, (3) heating the dried film above the glass transition temperature of the polymer, and (4) re-dispersion and chemical etching of the inorganic medium. The primary driver is capillary imbibition of the polymer melt into the inorganic colloidal template. In addition, 2D particle tracking experiments of dispersed rough particles in water were performed to probe the diffusional behaviour of the roughened objects in comparison with their smooth precursors. We show that, despite large scale roughness (up to 10% asperity size with respect to particle diameter), Stokes law is obeyed and the particle motion can be modelled simply with the Stokes-Einstein-Sutherland relation.

Mechanistic Insight into the Synthesis of Silica-Based "Matchstick" Colloids.

We report an insight into the synthesis of silica-based "matchstick"-shaped colloidal particles, which are of interest in the area of self-propulsion on small length scales. The generation of aqueous emulsion droplets dispersed in an n-pentanol-rich continuous phase and their use as reaction centers allows for the fabrication of siliceous microparticles that exhibit anisotropy in both particle morphology, that is, a "matchstick" shape, and chemistry, that is, a transition-metal oxide-enriched head. We provide a series of kinetic studies to gain a mechanistic understanding and unravel the particle formation and growth processes. Additionally, we demonstrate the ability to select the aspect ratio of the "matchstick" particle in a straightforward manner.