Synthesis and cell-free cloning of DNA libraries using programmable microfluidics.

Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development.

The dynamics and stability of lubricating oil films during droplet transport by electrowetting in microfluidic devices.

The operation of digital microfluidic devices with water droplets manipulated by electrowetting is critically dependent on the static and dynamic stability and lubrication properties of the oil films that separate the droplets from the solid surfaces. The factors determining the stability of the films and preventing surface fouling in such systems are not yet thoroughly understood and were experimentally investigated in this study. The experiments were performed using a standard digital microfluidic cartridge in which water droplets enclosed in a thin, oil-filled gap were transported over an array of electrodes. Stable, continuous oil films separated the droplets from the surfaces when the droplets were stationary. During droplet transport, capillary waves formed in the films on the electrode surfaces as the oil menisci receded. The waves evolved into dome-shaped oil lenses. Droplet deformation and oil displacement caused the films at the surface opposite the electrode array to transform into dimples of oil trapped over the centers of the droplets. Lower actuation voltages were associated with slower film thinning and formation of fewer, but larger, oil lenses. Lower ac frequencies induced oscillations in the droplets that caused the films to rupture. Films were also destabilized by addition of surfactants to the oil or droplet phases. Such a comprehensive understanding of the oil film behavior will enable more robust electrowetting-actuated lab-on-a-chip devices through prevention of loss of species from droplets and contamination of surfaces at points where films may break.

Macro to microfluidics system for biological environmental monitoring.

Biological environmental monitoring (BEM) is a growing field of research which challenges both microfluidics and system automation. The aim is to develop a transportable system with analysis throughput which satisfies the requirements: (i) fully autonomous, (ii) complete protocol integration from sample collection to final analysis, (iii) detection of diluted molecules or biological species in a large real life environmental sample volume, (iv) robustness and (v) flexibility and versatility. This paper discusses all these specifications in order to define an original fluidic architecture based on three connected modules, a sampling module, a sample preparation module and a detection module. The sample preparation module highly concentrates on the pathogens present in a few mL samples of complex and unknown solutions and purifies the pathogens' nucleic acids into a few µL of a controlled buffer. To do so, a two-step concentration protocol based on magnetic beads is automated in a reusable macro-to-micro fluidic system. The detection module is a PCR based miniaturized platform using digital microfluidics, where reactions are performed in 64 nL droplets handled by electrowetting on dielectric (EWOD) actuation. The design and manufacture of the two modules are reported as well as their respective performances. To demonstrate the integration of the complete protocol in the same system, first results of pathogen detection are shown.