Jean-Christophe Baret

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.

Droplet-Based Microfluidic Platforms for the Encapsulation and Screening of Mammalian Cells and Multicellular Organisms

High-throughput, cell-based assays require small sample volumes to reduce assay costs and to allow for rapid sample manipulation. However, further miniaturization of conventional microtiter plate technology is problematic due to evaporation and capillary action. To overcome these limitations, we describe droplet-based microfluidic platforms in which cells are grown in aqueous microcompartments separated by an inert perfluorocarbon carrier oil. Synthesis of biocompatible surfactants and identification of gas-permeable storage systems allowed human cells, and even a multicellular organism (C. elegans), to survive and proliferate within the microcompartments for several days. Microcompartments containing single cells could be reinjected into a microfluidic device after incubation to measure expression of a reporter gene. This should open the way for high-throughput, cell-based screening that can use >1000-fold smaller assay volumes and has approximately 500x higher throughput than conventional microtiter plate assays.

High-resolution dose–response screening using droplet-based microfluidics

A critical early step in drug discovery is the screening of a chemical library. Typically, promising compounds are identified in a primary screen and then more fully characterized in a dose–response analysis with 7–10 data points per compound. Here, we describe a robust microfluidic approach that increases the number of data points to approximately 10,000 per compound. The system exploits Taylor–Aris dispersion to create concentration gradients, which are then segmented into picoliter microreactors by droplet-based microfluidics. The large number of data points results in IC50 values that are highly precise (± 2.40% at 95% confidence) and highly reproducible (CV = 2.45%, n = 16). In addition, the high resolution of the data reveals complex dose–response relationships unambiguously. We used this system to screen a chemical library of 704 compounds against protein tyrosine phosphatase 1B, a diabetes, obesity, and cancer target. We identified a number of novel inhibitors, the most potent being sodium cefsulodine, which has an IC50 of 27 ± 0.83 μM.

Kinetic aspects of emulsion stabilization by surfactants: a microfluidic analysis.

In classical emulsification processes, surfactants play two roles: first, they reduce the interfacial tension, facilitating droplet deformation and rupture, and second, they reduce droplet coalescence. Here, we use a microfluidic emulsification system to completely uncouple these two processes, allowing stabilization against coalescence to be studied quantitatively and independently of droplet formation. We demonstrate that, in addition to the classical effect of stabilization by an increase of surfactant concentration, the dynamics of adsorption of surfactant at the water-oil interface is a key element for droplet stabilization. Microfluidic emulsification devices can therefore be tailored to improve emulsification while decreasing the concentration of surfactant by increasing the time before the droplets first come into contact.

Microfluidic mixing through electrowetting-induced droplet oscillations

We used electrowetting to trigger self-excited oscillations of millimeter-sized sessile droplets of water-glycerol mixtures in a viscosity range from 1 to 65 mPa s. During the oscillations the contact angle of the droplets varied periodically between [approximate]130° and 80° with a frequency between 10 and 125 s–1, depending on the viscosity and the drop size. By initially staining drops partially with fluorescent dye, we found that the liquid within the drop is completely mixed within 100–2000 oscillation cycles for low and high viscosities, respectively. Compared to pure diffusion, droplet oscillations accelerated mixing by approximately two orders of magnitude for millimeter-sized droplets

Quantitative Cell-Based Reporter Gene Assays Using Droplet-Based Microfluidics

We used a droplet-based microfluidic system to perform a quantitative cell-based reporter gene assay for a nuclear receptor ligand. Single Bombyx mori cells are compartmentalized in nanoliter droplets which function as microreactors with a >1000-fold smaller volume than a microtiter-plate well, together with eight or ten discrete concentrations of 20-hydroxyecdysone, generated by on-chip dilution over 3 decades and encoded by a fluorescent label. The simultaneous measurement of the expression of green fluorescent protein by the reporter gene and of the fluorescent label allows construction of the dose-response profile of the hormone at the single-cell level. Screening approximately 7500 cells per concentration provides statistically relevant data that allow precise measurement of the EC(50) (70 nM +/- 12%, alpha = 0.05), in agreement with standard methods as well as with literature data.

Gravity-driven flows of viscous liquids over two-dimensional topographies

Using phase-stepped interferometry, we have measured full two-dimensional maps of the free-surface shape of a thin liquid film of water flowing over an inclined plate with topography. The measurement technique allows us to image automatically the shape of the free surface in a single field of view of about 2.4 by 1.8 mm, with a lateral resolution of 3.1 μm and a height resolution of 0.3 μm. By imaging neighbouring regions and combining them, complete two-dimensional free-surface profiles of gravity-driven liquid films with a thickness ranging between 80 and 120 μm are measured, over step, trench, rectangular and square topographies with depths of 10 and 20 μm, and lateral dimensions of the order of 1 to several mm. The experimental results for both one- and two-dimensional flows are found to be in good agreement with existing models, including a recent two-dimensional Greens function of the linearized problem by Hayes et al. This extends the applicability of simple models to cases with a high value of topography steepness and low-viscosity liquids as in our experiments. A corollary of the agreement with the linear two-dimensional model is that our experimental results behave linearly, a convenient property that allows the free-surface response to complex topographies to be worked out from knowledge of the response to an elementary topography like a square.

High-Throughput Screening of Enzymes by Retroviral Display Using Droplet-Based Microfluidics

During the last 25 years, display techniques such as phage display have become very powerful tools for protein engineering, especially for the selection of monoclonal antibodies. However, while this method is extremely efficient for affinity-based selections, its use for the selection and directed evolution of enzymes is still very restricted. Furthermore, phage display is not suited for the engineering of mammalian proteins that require posttranslational modifications such as glycosylation or membrane anchoring. To circumvent these limitations, we have developed a system in which structurally complex mammalian enzymes are displayed on the surface of retroviruses and encapsulated into droplets of a water-in-oil emulsion. These droplets are made and manipulated using microfluidic devices and each droplet serves as an independent reaction vessel. Compartmentalization of single retroviral particles in droplets allows efficient coupling of genotype and phenotype. Using tissue plasminogen activator (tPA) as a model enzyme, we show that, by monitoring the enzymatic reaction in each droplet (by fluorescence), quantitative measurement of tPA activity in the presence of different concentrations of the endogenous inhibitor PAI-1 can be made on-chip. On-chip fluorescence-activated droplet sorting allowed the processing of 500 samples per second and the specific collection of retroviruses displaying active wild-type tPA from a model library with a 1000-fold excess of retroviruses displaying a non-active control enzyme. During a single selection cycle, a more than 1300-fold enrichment of the active wild-type enzyme was demonstrated.

Dynamics of molecular transport by surfactants in emulsions

We consider the dynamics of equilibration of the chemical potential of a fluorophore in a monodisperse emulsion containing droplets with two initially different concentrations of the fluorophore. Although the exchange mechanism involves a single timescale at the droplet (microscopic) level, the organisation of the droplets determines the exchange dynamics at the population (macroscopic) level. The micelle concentration in the continuous phase and the chemistry of the fluorophore control the microscopic exchange rate while the disorder of the initial condition determines the power-law of the long timescale, recovered in a minimal analytical model. We also show here that an additive in the droplet such as Bovine Serum Albumin (BSA) acts on the microscopic exchange rate and slows down the exchange process by increasing the solubility of the fluorophore in the dispersed phase rather than by creating a viscoelastic layer at the droplet interface.

Extremal model for amorphous media plasticity.

An extremal model for the plasticity of amorphous materials is studied in a simple two-dimensional antiplane geometry. The steady state is analyzed through numerical simulations. Long-range spatial and temporal correlations in local slip events are shown to develop, leading to nontrivial and highly anisotropic scaling laws. In particular, the plastic strain is shown to concentrate statistically over a region which tends to align perpendicular to the displacement gradient. By construction, the model can be seen as giving rise to a depinning transition, the threshold of which (i.e., the macroscopic yield stress) also reveals scaling properties reflecting the localization of the activity.