MP08:Kaminski:Automated microfluidic system for rapid generation of subnanoliter droplets with predefined composition

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Tomasz Kamiński*, Sławomir Jakieła*, Krzysztof Churski* and Piotr Garstecki*
* Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, POLAND

We demonstrate a system for rapid and automated generation of libraries of subnanoliter droplets from sequences of microliter droplets of arbitrary compositions. This system forms a useful link between the automated systems for high-throughput generation of reaction conditions and those for high-throughput screening of random libraries.
Droplet microfluidics challenges the classical robotic stations in high-throughput screening applications, offering smaller reaction volumes and up to thousands of repetitions while reducing the cost and time of the protocols. Still, several challenges in droplet microfluidic remain to be tackled. One hurdle is in the lack of facile methods for rapid and automated generation of libraries of nL droplets containing a range of concentrations and combinations of the chemistry to be tested in a high-throughput screen.
We tested various devices fabricated in polycarbonate or PDMS and uses controlled electromagnetic external valves. This allows for complete synchronization of all the processes, including formation of parent droplets, rapid (> hundreds of Hz) splitting of these droplets in the flow focusing module and introduction of gaseous spacers between the families of nL droplets. The aqueous droplets are stable against coalescence in the continuous liquid (3% PFPE-PEG-PFPE triblock surfactant in fluorocarbon oil).
We first generate droplets on-demand from three sources of solutions, merge these droplets into ~ 1 microliter mixtures. These parental plugs are subsequently injected either on the same chip, or onto a second chip via a tubing connector where they are fragmented in a flow-focusing module into thousands of daughter monodisperse droplets of subnanoliter volumes (~0.5 nL). We have tested dozens of geometries of the chip and found that in order to minimize the dispersion of the volumes of these daughter droplets it is necessary to gradually decrease the width of the microfluidic channel upstream of the FF module and that the whole parent droplet must be squeezed into the section of a narrow channel before it enters the orifice of the FF splitter. This procedure omits the problems associated with the changes of the curvature of the parent droplet and thus diminishes influence of Laplacian pressure on the process of formation of daughter droplets. Moreover, we investigated role of surfactant during the droplet fragmentation and optimized its concentration, in order to achieve monodisperse libraries of droplets (CV of volume<3%).
The system that we describe shows the complementation of the automated systems for generation of large (i.e. ~ 1 microliter) plugs of arbitrary composition with the methods for high-throughput screening of small (i.e. ~ nL) droplet libraries. Our device can serve as a crucial improvement in the single cell or single molecule microfluidic screening assays and droplet digital PCR systems. Additionally this systems can be used in environmental microbiology and in directed evolution of enzymes.

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