Droplet microfluidic technology for single-cell high-throughput screening.

We present a droplet-based microfluidic technology that enables high-throughput screening of single mammalian cells. This integrated platform allows for the encapsulation of single cells and reagents in independent aqueous microdroplets (1 pL to 10 nL volumes) dispersed in an immiscible carrier oil and enables the digital manipulation of these reactors at a very high-throughput. Here, we validate a full droplet screening workflow by conducting a droplet-based cytotoxicity screen. To perform this screen, we first developed a droplet viability assay that permits the quantitative scoring of cell viability and growth within intact droplets. Next, we demonstrated the high viability of encapsulated human monocytic U937 cells over a period of 4 days. Finally, we developed an optically-coded droplet library enabling the identification of the droplets composition during the assay read-out. Using the integrated droplet technology, we screened a drug library for its cytotoxic effect against U937 cells. Taken together our droplet microfluidic platform is modular, robust, uses no moving parts, and has a wide range of potential applications including high-throughput single-cell analyses, combinatorial screening, and facilitating small sample analyses.

A Droplet Microfluidics Based Platform for Mining Metagenomic Libraries for Natural Compounds.

Historically, microbes from the environment have been a reliable source for novel bio-active compounds. Cloning and expression of metagenomic DNA in heterologous strains of bacteria has broadened the range of potential compounds accessible. However, such metagenomic libraries have been under-exploited for applications in mammalian cells because of a lack of integrated methods. We present an innovative platform to systematically mine natural resources for pro-apoptotic compounds that relies on the combination of bacterial delivery and droplet microfluidics. Using the violacein operon from C. violaceum as a model, we demonstrate that E. coli modified to be invasive can serve as an efficient delivery vehicle of natural compounds. This approach permits the seamless screening of metagenomic libraries with mammalian cell assays and alleviates the need for laborious extraction of natural compounds. In addition, we leverage the unique properties of droplet microfluidics to amplify bacterial clones and perform clonal screening at high-throughput in place of one-compound-per-well assays in multi-well format. We also use droplet microfluidics to establish a cell aggregate strategy that overcomes the issue of background apoptosis. Altogether, this work forms the foundation of a versatile platform to efficiently mine the metagenome for compounds with therapeutic potential.

Phase dependent electrochemical properties of polar self-assembled monolayers (SAMs) modified via the fusion of mixed phospholipid vesicles.

Unilamellar vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and varying quantities of either 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol) (sodium salt) (DMPG) or 1,2-dimyristoyl-3-trimethylammonium-propane (chloride salt) (DMTAP) were used to deposit lipid bilayer assemblies on self-assembled monolayers (SAMs) on gold. The supporting SAMs in turn were composed of ferrocene-functionalized hexadecanethiol chains (FcC16SH) diluted to low coverage in 1-hydroxylhexadecanethiol (HOC16SH) or a single-component monolayer phase of the latter. The mass coverages of the DMPC/DMPG layers deposited in this way were measured using surface plasmon resonance (SPR) and found to decrease with an increasing content of DMPG in the vesicles. The SPR data show that the lipid assembly, while stable with respect to gentle rinsing in aqueous buffer, is reversible and the lipid adlayer is removable by immersion in a solvent such as ethanol. The effects of the adsorbed lipid layer on the electrochemical interactions of the hybrid lipid/SAM with several redox probes [e.g., K4Fe(CN)6, Ru(NH3)6Cl3, and CsHsFe-[(C5H4CH2N+H(CH3)2] were characterized using cyclic voltammetry (CV). At a composition of 5% DMPG in DMPC, the permeabilities of the probes through the lipid layer were affected significantly relative to that observed with a pure DMPC layer. These effects include a striking observation of an enhanced, ionic-charge-specific molecular discrimination of the electrochemical probes. At higher concentrations of the DMPG, significant permeation of the lipid adlayer was seen for all the probes. These latter changes are also attended by a significant increase in the capacitive currents measured in CV experiments as compared to those observed for either a pure SAM or one modified by only DMPC. This effect likely results from the influence of the charged lipid on the diffuse Gouy-Chapman electrolyte layer at the SAM interface. In contrast to the behaviors seen with DMPG, the incorporation of DMTAP into the adsorbed DMPC had no impact on the permeation of the adlayer by soluble redox probes as judged by the observed electrochemistry, a result that appears to correlate with a less ideal mixing of lipids in the DMPC/DMTAP system relative to that of a DMPC/DMPG mixture.