We have developed a new hybrid droplet-to-digital microfluidic platform (D2D) that integrates droplet-in-channel microfluidics with digital microfluidics (DMF) for performing multi-step assays. This D2D platform combines the strengths of the two formats-droplets-in-channel for facile generation of droplets containing single cells, and DMF for on-demand manipulation of droplets including control of different droplet volumes (pL-μL), creation of a dilution series of ionic liquid (IL), and parallel single cell culturing and analysis for IL toxicity screening. This D2D device also allows for automated analysis that includes a feedback-controlled system for merging and splitting of droplets to add reagents, an integrated Peltier element for parallel cell culture at optimum temperature, and an impedance sensing mechanism to control the flow rate for droplet generation and preventing droplet evaporation. Droplet-in-channel is well-suited for encapsulation of single cells as it allows the careful manipulation of flow rates of aqueous phase containing cells and oil to optimize encapsulation. Once single cell containing droplets are generated, they are transferred to a DMF chip via a capillary where they are merged with droplets containing IL and cultured at 30 °C. The DMF chip, in addition to permitting cell culture and reagent (ionic liquid/salt) addition, also allows recovery of individual droplets for off-chip analysis such as further culturing and measurement of ethanol production. The D2D chip was used to evaluate the effect of IL/salt type (four types: NaOAc, NaCl, [C2mim] [OAc], [C2mim] [Cl]) and concentration (four concentrations: 0, 37.5, 75, 150 mM) on the growth kinetics and ethanol production of yeast and as expected, increasing IL concentration led to lower biomass and ethanol production. Specifically, [C2mim] [OAc] had inhibitory effects on yeast growth at concentrations 75 and 150 mM and significantly reduced their ethanol production compared to cells grown in other ILs/salts. The growth curve trends obtained by D2D matched conventional yeast culturing in microtiter wells, validating the D2D platform. We believe that our approach represents a generic platform for multi-step biochemical assays such as drug screening, digital PCR, enzyme assays, immunoassays and cell-based assays.

We present a water-in-oil droplet microfluidic platform for transformation, culture and expression of recombinant proteins in multiple host organisms including bacteria, yeast and fungi. The platform consists of a hybrid digital microfluidic/ channel-based droplet chip with integrated temperature control to allow complete automation and integration of plasmid addition, heat-shock transformation, addition of selection medium, culture, and protein expression. The microfluidic format permitted significant reduction in consumption (100-fold) of expensive reagents such as DNA and enzymes compared to the benchtop method. The chip contains a channel to continuously replenish oil to the culture chamber to provide a fresh supply of oxygen to the cells for long-term (∼5 days) cell culture. The flow channel also replenished oil lost to evaporation and increased the number of droplets that could be processed and cultured. The platform was validated by transforming several plasmids into Escherichia coli including plasmids containing genes for fluorescent proteins GFP, BFP and RFP; plasmids with selectable markers for ampicillin or kanamycin resistance; and a Golden Gate DNA assembly reaction. We also demonstrate the applicability of this platform for transformation in widely used eukaryotic organisms such as Saccharomyces cerevisiae and Aspergillus niger. Duration and temperatures of the microfluidic heat-shock procedures were optimized to yield transformation efficiencies comparable to those obtained by benchtop methods with a throughput up to 6 droplets/min. The proposed platform offers potential for automation of molecular biology experiments significantly reducing cost, time and variability while improving throughput.