Jeremy Agresti

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.

Biocompatible surfactants for water-in-fluorocarbon emulsions

Drops of water-in-fluorocarbon emulsions have great potential for compartmentalizing both in vitro and in vivo biological systems; however, surfactants to stabilize such emulsions are scarce. Here we present a novel class of fluorosurfactants that we synthesize by coupling oligomeric perfluorinated polyethers (PFPE) with polyethyleneglycol (PEG). We demonstrate that these block copolymer surfactants stabilize water-in-fluorocarbon oil emulsions during all necessary steps of a drop-based experiment including drop formation, incubation, and reinjection into a second microfluidic device. Furthermore, we show that aqueous drops stabilized with these surfactants can be used for in vitro translation (IVT), as well as encapsulation and incubation of single cells. The compatability of this emulsion system with both biological systems and polydimethylsiloxane (PDMS) microfluidic devices makes these surfactants ideal for a broad range of high-throughput, drop-based applications.

Designer emulsions using microfluidics

We describe new developments for the controlled fabrication of monodisperse emulsions using microfluidics. We use glass capillary devices to generate single, double, and higher order emulsions with exceptional precision. These emulsions can serve as ideal templates for generating well-defined particles and functional vesicles. Polydimethylsiloxane microfluidic devices are also used to generate picoliter-scale water-in-oil emulsions at rates as high as 10 000 drops per second. These emulsions have great potential as individual microvessels in high-throughput screening applications, where each drop serves to encapsulate single cells, genes, or reactants.

Drop-based microfluidic devices for encapsulation of single cells

We use microfluidic devices to encapsulate, incubate, and manipulate individual cells in picoliter aqueous drops in a carrier fluid at rates of up to several hundred Hz. We use a modular approach with individual devices for each function, thereby significantly increasing the robustness of our system and making it highly flexible and adaptable to a variety of cell-based assays. The small volumes of the drops enables the concentrations of secreted molecules to rapidly attain detectable levels. We show that single hybridoma cells in 33 pL drops secrete detectable concentrations of antibodies in only 6 h and remain fully viable. These devices hold the promise of developing microfluidic cell cytometers and cell sorters with much greater functionality, allowing assays to be performed on individual cells in their own microenvironment prior to analysis and sorting.

High-throughput injection with microfluidics using picoinjectors

Adding reagents to drops is one of the most important functions in droplet-based microfluidic systems; however, a robust technique to accomplish this does not exist. Here, we introduce the picoinjector, a robust device to add controlled volumes of reagent using electro-microfluidics at kilohertz rates. It can also perform multiple injections for serial and combinatorial additions.

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.

Directed evolution by in vitro compartmentalization

aqueous droplets per ml of emulsion. The majority of droplets contain no more than a single gene along with all of the molecular machinery needed to express that gene. The expressed proteins and the products of their catalytic activities cannot leave the droplets, and so genotype is coupled to phenotype

A multi-color fast-switching microfluidic droplet dye laser

We describe a multi-color microfluidic dye laser operating in whispering gallery mode based on a train of alternating droplets containing solutions of different dyes; this laser is capable of switching the wavelength of its emission between 580 nm and 680 nm at frequencies up to 3.6 kHz-the fastest among all dye lasers reported; it has potential applications in on-chip spectroscopy and flow cytometry.

Fabrication of monodisperse thermosensitive microgels and gel capsules in microfluidic devices

We use droplet-based microfluidic techniques to produce monodisperse poly(N-isopropylacrylamide) gel particles in the size range of 10–1000 µm. Our techniques offer exquisite control over both outer dimensions and inner morphology of the particles. We demonstrate this control by fabricating conventional microgels, microgels with embedded materials and voids, and gel microcapsules with single- and multi-phase cores. These techniques should be applicable for the synthesis of particles and capsules of a variety of chemical compositions and for the generation of higher order “supraparticles” by directed assembly of colloidal particles in droplets.

Tracking lineages of single cells in lines using a microfluidic device

Cells within a genetically identical population exhibit phenotypic variation that in some cases can persist across multiple generations. However, information about the temporal variation and familial dependence of protein levels remains hidden when studying the population as an ensemble. To correlate phenotypes with the age and genealogy of single cells over time, we developed a microfluidic device that enables us to track multiple lineages in parallel by trapping single cells and constraining them to grow in lines for as many as 8 divisions. To illustrate the utility of this method, we investigate lineages of cells expressing one of 3 naturally regulated proteins, each with a different representative expression behavior. Within lineages deriving from single cells, we observe genealogically related clusters of cells with similar phenotype; cluster sizes vary markedly among the 3 proteins, suggesting that the time scale of phenotypic persistence is protein-specific. Growing lines of cells also allows us to dynamically track temporal fluctuations in protein levels at the same time as pedigree relationships among the cells as they divide in the chambers. We observe bursts in expression levels of the heat shock protein Hsp12-GFP that occur simultaneously in mother and daughter cells. In contrast, the ribosomal protein Rps8b-GFP shows relatively constant levels of expression over time. This method is an essential step toward understanding the time scales of phenotypic variation and correlations in phenotype among single cells within a population.