Andrew S. Utada

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.

Living in the matrix: assembly and control of Vibrio cholerae biofilms

Nearly all bacteria form biofilms as a strategy for survival and persistence. Biofilms are associated with biotic and abiotic surfaces and are composed of aggregates of cells that are encased by a self-produced or acquired extracellular matrix. Vibrio cholerae has been studied as a model organism for understanding biofilm formation in environmental pathogens, as it spends much of its life cycle outside of the human host in the aquatic environment. Given the important role of biofilm formation in the V. cholerae life cycle, the molecular mechanisms underlying this process and the signals that trigger biofilm assembly or dispersal have been areas of intense investigation over the past 20 years. In this Review, we discuss V. cholerae surface attachment, various matrix components and the regulatory networks controlling biofilm formation.

25th Anniversary Article: Double Emulsion Templated Solid Microcapsules: Mechanics And Controlled Release

How droplet microfluidics can be used to fabricate solid-shelled microcapsules having precisely controlled release behavior is described. Glass capillary devices enable the production of monodisperse double emulsion drops, which can then be used as templates for microcapsule formation. The exquisite control afforded by microfluidics can be used to tune the compositions and geometrical characteristics of the microcapsules with exceptional precision. The use of this approach to fabricate microcapsules that only release their contents when exposed to a specific stimulus--such as a change in temperature, exposure to light, a change in the chemical environment, or an external stress--only after a prescribed time delay, and at a prescribed rate is reviewed.

Encapsulating bacteria in agarose microparticles using microfluidics for high-throughput cell analysis and isolation

The high-throughput analysis and isolation of bacterial cells encapsulated in agarose microparticles using fluorescence-activated cell sorting (FACS) is described. Flow-focusing microfluidic systems were used to create monodisperse microparticles that were ∼30 μm in diameter. The dimensions of these particles made them compatible with flow cytometry and FACS, and the sensitivity of these techniques reduced the incubation time for cell replication before analyses were carried out. The small volume of the microparticles (∼1-50 pL) minimized the quantity of reagents needed for bacterial studies. This platform made it possible to screen and isolate bacteria and apply a combination of techniques to rapidly determine the target of biologically active small molecules. As a pilot study, Escherichia coli cells were encapsulated in agarose microparticles, incubated in the presence of varying concentrations of rifampicin, and analyzed using FACS. The minimum inhibitory concentration of rifampicin was determined, and spontaneous mutants that had developed resistance to the antibiotic were isolated via FACS and characterized by DNA sequencing. The β-subunit of RNA polymerase, RpoB, was confirmed as the target of rifampicin, and Q513L was the mutation most frequently observed. Using this approach, the time and quantity of antibiotics required for the isolation of mutants was reduced by 8- and 150-fold, respectively, compared to conventional microbiological techniques using nutrient agar plates. We envision that this technique will have an important impact on research in chemical biology, natural products chemistry, and the discovery and characterization of biologically active secondary metabolites.

Synthesis of Monodisperse Microparticles from Non-Newtonian Polymer Solutions with Microfluidic Devices

Microfluidic devices can form emulsions that are highly uniform in size;[1–3] they can also form compound emulsions, in which each supradroplet contains exactly the same number of internal droplets, packed in exactly the same configuration.[4–6] Because the drops can be formed with a highly controlled structure and uniformity, they are useful as templates to synthesize monodisperse particles. In such a process, microfluidic devices are used to form droplets with the desired structure, which are then solidified to produce particles. This allows synthesis of particles with a variety of shapes, including Janus particles, nonspherical dimers, and core–shell capsules.[7–10] However, the fluid precursors must be compatible with the formation of drops in microfluidic devices: this precondition limits the applicability of this technique. For example, this circumstance requires fluids with a low viscosity, negligible viscoelastic response, and moderate interfacial tension. If even one of these constraints is not met, it is difficult to achieve drop formation in the stable dripping regime. Instead, jetting occurs, resulting in the production of polydisperse particles.[11] This represents a significant limitation, as the most useful materials for making particles typically have properties that do not meet these constraints. For example, lipid melts, due to their amphiphilic chemical properties, tend to be viscous and have low interfacial tension with oil or aqueous carrier phases. Solutions of long-chain polymers like poly(N-isopropylacrylamide) (pNIPAM)[12] or polyurethane–polybutadienediol (pU–pBDO),[13] form excellent particles, but tend to be viscous and have significant viscoelastic response at the shear rates needed for controlled drop formation.[1,11,14] As a consequence, these fluids, and many others like them, cannot be used to synthesize particles in microfluidic devices, greatly limiting the applicability of this technique. To overcome the limitations with such fluids, a more robust drop formation mechanism is needed.

C-di-GMP Regulates Motile to Sessile Transition by Modulating MshA Pili Biogenesis and Near-Surface Motility Behavior in Vibrio cholerae

In many bacteria, including Vibrio cholerae, cyclic dimeric guanosine monophosphate (c-di-GMP) controls the motile to biofilm life style switch. Yet, little is known about how this occurs. In this study, we report that changes in c-di-GMP concentration impact the biosynthesis of the MshA pili, resulting in altered motility and biofilm phenotypes in V. cholerae. Previously, we reported that cdgJ encodes a c-di-GMP phosphodiesterase and a ΔcdgJ mutant has reduced motility and enhanced biofilm formation. Here we show that loss of the genes required for the mannose-sensitive hemagglutinin (MshA) pilus biogenesis restores motility in the ΔcdgJ mutant. Mutations of the predicted ATPase proteins mshE or pilT, responsible for polymerizing and depolymerizing MshA pili, impair near surface motility behavior and initial surface attachment dynamics. A ΔcdgJ mutant has enhanced surface attachment, while the ΔcdgJmshA mutant phenocopies the high motility and low attachment phenotypes observed in a ΔmshA strain. Elevated concentrations of c-di-GMP enhance surface MshA pilus production. MshE, but not PilT binds c-di-GMP directly, establishing a mechanism for c-di-GMP signaling input in MshA pilus production. Collectively, our results suggest that the dynamic nature of the MshA pilus established by the assembly and disassembly of pilin subunits is essential for transition from the motile to sessile lifestyle and that c-di-GMP affects MshA pilus assembly and function through direct interactions with the MshE ATPase.