Adam R. Abate

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.

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.

Janus Particles Templated from Double Emulsion Droplets Generated Using Microfluidics

We present a simple microfluidics-based technique to fabricate Janus particles using double-emulsion droplets as templates. Since each half of the particles is templated from a different immiscible fluid, this method enables the formation of particles from two materials with vastly different properties. The use of microfluidics affords excellent control over the size, morphology, and monodispersity of the particles.

High-order multiple emulsions formed in poly(dimethylsiloxane) microfluidics.

Multiple emulsions are nested sets of drops. Drops of one kind of fluid are encapsulated inside drops of a second fluid, which themselves can be encapsulated inside drops of yet another fluid. Such ‘‘emulsions within emulsions’’ are very useful for many applications, including for the encapsulation of actives, such as nutrients, pesticides, drugs, and detergents, and as templates for the synthesis of colloids with structured interiors. Multiple emulsions are typically made in bulk using shear cells or porous membrane plates. To form the multiple emulsions, the drops from one emulsification are fed back into the apparatus with additional fluids and are emulsified again. Such methods can form large quantities of drops very rapidly; however, the individual drop formation is poorly controlled. This produces some double emulsions but they are exceedingly polydisperse. For higher-order multiple emulsions, such as triple emulsions or quadruple emulsions, the excessive polydispersity makes the methods impractical. With glass microcapillary devices, monodisperse multiple emulsions can be formed with controlled structure, albeit in much smaller quantities. As opposed to bulk methods in which many drops are formed in parallel with little control, in microcapillary devices, drops are formed individually with a high degree of control. However, the glass devices are difficult to fabricate, requiring the hand alignment of separate microcapillary tubes. This is labor intensive and makes the devices difficult to scale up sequentially for producing higherorder multiple emulsions or in parallel for producing larger quantities. A superior method would combine the control of microfluidic drop formation with increased scalability. In this paper, we present a simple system to form monodisperse high-order multiple emulsions. This method combines the control of microfluidic drop formation with the

Droplet Microfluidics for Fabrication of Non-Spherical Particles

We describe new developments for controlled fabrication of monodisperse non-spherical particles using droplet microfluidics. The high degree of control afforded by microfluidic technologies enables generation of single and multiple emulsion droplets. We show that these droplets can be transformed to non-spherical particles through further simple, spontaneous processing steps, including arrested coalescence, asymmetric polymer solidification, polymerization in microfluidic flow, and evaporation-driven clustering. These versatile and scalable microfluidic approaches can be used for producing large quantities of non-spherical particles that are monodisperse in both size and shape; these have great potential for commercial applications.

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.

Smart Microgel Capsules from Macromolecular Precursors

Microgel particles and capsules which consist of multiple layers can be fabricated using droplet microfluidics, but in existing methods, emulsion templating forms layers of dissimilar polarity. In this paper, we fabricate functional microgel capsules that consist of two miscible yet distinct layers. We use microfluidic devices to template micrometer-sized drops that are loaded with prepolymerized precursors and solidify them through a polymer-analogous reaction. This allows the particle morphology to be controlled and prevents pronounced interpenetration of the different layers despite their miscibility. We use polyacrylamide and poly(N-isopropylacrylamide) precursors to form thermoresponsive core-shell microparticles and demonstrate their utility for encapsulation and controlled release applications.

Ultrahigh-Throughput Mammalian Single-Cell Reverse-Transcriptase Polymerase Chain Reaction in Microfluidic Drops

The behaviors of complex biological systems are often dictated by the properties of their heterogeneous and sometimes rare cellular constituents. Correspondingly, the analysis of individual cells from a heterogeneous population can reveal information not obtainable by ensemble measurements. Reverse-transcriptase polymerase chain reaction (RT-PCR) is a widely used method that enables transcriptional profiling and sequencing analysis on bulk populations of cells. Major barriers to successfully implementing this technique for mammalian single-cell studies are the labor, cost, and low-throughput associated with current approaches. In this report, we describe a novel droplet-based microfluidic system for performing ~50000 single-cell RT-PCR reactions in a single experiment while consuming a minimal amount of reagent. Using cell type-specific staining and TaqMan RT-PCR probes, we demonstrate the identification of specific cells from a mixed human cell population. The throughput, robust detection rate and specificity of this method makes it well-suited for characterizing large, heterogeneous populations of cells at the transcriptional level.

Beating Poisson encapsulation statistics using close-packed ordering†

Loading drops with discrete objects, such as particles and cells, is often necessary when performing chemical and biological assays in microfluidic devices. However, random loading techniques are inefficient, yielding a majority of empty and unusable drops. We use deformable particles that are close packed to insert a controllable number of particles into every drop. This provides a simple, flexible means of efficiently encapsulating a controllable number of particles per drop.

Microfluidic sorting with high-speed single-layer membrane valves

Sorting is one of the most important applications of microfluidic devices; however, current sorters place specific requirements on the density, size, and electrical properties of the objects to be sorted, limiting applicability. We present widely applicable microfluidic sorting. We use high-speed single-layer membrane valves to control flows in a bifurcating channel junction, to direct the paths of objects. This allows sorting at hundreds of hertz. Moreover, since the sorting action is mechanical, it is very widely applicable—to drops, particles, and even living cells.