Keunho Ahn

Ultrahigh-throughput screening in drop-based microfluidics for directed evolution

The explosive growth in our knowledge of genomes, proteomes, and metabolomes is driving ever-increasing fundamental understanding of the biochemistry of life, enabling qualitatively new studies of complex biological systems and their evolution. This knowledge also drives modern biotechnologies, such as molecular engineering and synthetic biology, which have enormous potential to address urgent problems, including developing potent new drugs and providing environmentally friendly energy. Many of these studies, however, are ultimately limited by their need for even-higher-throughput measurements of biochemical reactions. We present a general ultrahigh-throughput screening platform using drop-based microfluidics that overcomes these limitations and revolutionizes both the scale and speed of screening. We use aqueous drops dispersed in oil as picoliter-volume reaction vessels and screen them at rates of thousands per second. To demonstrate its power, we apply the system to directed evolution, identifying new mutants of the enzyme horseradish peroxidase exhibiting catalytic rates more than 10 times faster than their parent, which is already a very efficient enzyme. We exploit the ultrahigh throughput to use an initial purifying selection that removes inactive mutants; we identify ∼100 variants comparable in activity to the parent from an initial population of ∼107. After a second generation of mutagenesis and high-stringency screening, we identify several significantly improved mutants, some approaching diffusion-limited efficiency. In total, we screen ∼108 individual enzyme reactions in only 10 h, using < 150 μL of total reagent volume; compared to state-of-the-art robotic screening systems, we perform the entire assay with a 1,000-fold increase in speed and a 1-million-fold reduction in cost.

Dielectrophoretic manipulation of drops for high-speed microfluidic sorting devices

We demonstrate a high-throughput drop sorter for microfluidic devices that uses dielectrophoretic forces. Microelectrodes underneath a polydimethylsiloxane channel produce forces of more than 10nN on a water drop in an inert oil, resulting in sorting rates greater than 1.6kHz. We investigate the dependence of such forces on drop size and flow. Alternate designs with electrodes on either side of a symmetric channel Y junction provide refined control over droplet selection.

Electrocoalescence of drops synchronized by size-dependent flow in microfluidic channels

The use of microfluidic devices to control drops of water in a carrier oil is a promising means of performing biological and chemical assays. An essential requirement for this is the controlled coalescence of pairs of drops to mix reagents together. We show that this can be accomplished through electrocoalescence of drops synchronized by size-dependent flow in microfluidic channels. Smaller drops move faster due to the Poiseuille flow, allowing pairs of surfactant-stabilized drops to be brought into contact where they are coalesced with an electric field. We apply this method to an enzyme assay to measure enzyme kinetic constants.