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We have developed a method for high-throughput isothermal amplification of single DNA molecules in a droplet-based microfluidic system. DNA amplification in droplets was analyzed using an intercalating fluorochrome, allowing fast and accurate 'digital' quantification of the template DNA based on the Poisson distribution of DNA molecules in droplets. The clonal amplified DNA in each 2 pL droplet was further analyzed by measuring the enzymatic activity of the encoded proteins after fusion with a 15 pL droplet containing an in vitro translation system.

This paper describes a method to control the volume and the velocity of drops generated in a flow-focusing device dynamically and independently. This method involves simultaneous tuning of the temperature of the nozzle of the device and of the flow rate of the continuous phase; the method requires a continuous phase liquid that has a viscosity that varies steeply with temperature. Increasing the temperature of the flow-focusing nozzle from 0 to 80 [degrees]C increased the volume of the drops by almost 2 orders of magnitude. Tuning both the temperature and the flow rate controlled the drop volume and the drop velocity independently; this feature is not possible in a basic flow-focusing device. This paper also demonstrates a procedure for identifying the range of possible drop volumes and drop velocities for a given flow-focusing device and shows how to generate drops with a specified volume and velocity within this range. This method is easy to implement in on-chip applications where thermal management is already incorporated in the system, such as DNA amplification using the polymerase chain reaction and nanoparticle synthesis.

Droplet-based microfluidics has raised a lot of interest recently due to its wide applications to screening biological/chemical assays with high throughput. Despite the advances on droplet-based assays involving cells, gene delivery methods that are compatible with the droplet platform have been lacking. In this report, we demonstrate a simple microfluidic device that encapsulates cells into aqueous droplets and then electroporates the encapsulated cells. The electroporation occurs when the cell-containing droplets (in oil) flow through a pair of microelectrodes with a constant voltage established in between. We investigate the parameters and characteristics of the electroporation. We demonstrate delivering enhanced green fluorescent protein (EGFP) plasmid into Chinese hamster ovary (CHO) cells. We envision the application of this technique to high-throughput functional genomics studies based on droplet microfluidics.

A microfluidic method to rapidly measure the octanol--water partition coefficient in thousands of individual picoliter drops is described. AT-junction microfluidic chip is used to generate a segmented flow of monodisperse, fluorescein-laden water in octanol carder fluid. The partitioning of individual drops reaches equilibrium in less than 2 s. Epifluorescence microscopy is used measure the partition coefficient of fluorescein as a function of pH. Results compare well with previous measurements using traditional shake-flask methods. The methods presented here are rapid, provide detailed statistics, and can be run in parallel, enabling the simultaneous partitioning of thousands of compounds for various applications such as drug development, environmental testing, and combinatorial chemistry. Microfluidic partitioning and extraction in picoliter drops may be useful for studying molecules and particles away from their equilibrium state and in cases with limited samples.

Rapid kinetic measurements are important in understanding chemical interactions especially for biological molecules. Herein, we present a droplet-based microfluidic platform to study streptavidin--biotin binding kinetics with millisecond time resolution. With integration of a confocal fluorescence detection system, individual droplets can be monitored and characterized online to extract kinetic information. Using this approach, binding kinetics between streptavidin and biotin were observed via fluorescence resonance energy transfer. The binding rate constant of streptavidin and biotin was found to be in a range of 3.0 x [10.sup.6] - 4.5 x [10.sup.7] [M.sup.-1] [s.sup.-1].