Abdeslam El Harrak

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.

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.

New generation of amino coumarin methyl sulfonate-based fluorogenic substrates for amidase assays in droplet-based microfluidic applications.

Droplet-based microfluidics is a powerful tool for biology and chemistry as it allows the production and the manipulation of picoliter-size droplets acting as individual reactors. In this format, high-sensitivity assays are typically based on fluorescence, so fluorophore exchange between droplets must be avoided. Fluorogenic substrates based on the coumarin leaving group are widely used to measure a variety of enzymatic activities, but their application in droplet-based microfluidic systems is severely impaired by the fast transport of the fluorescent product between compartments. Here we report the synthesis of new amidase fluorogenic substrates based on 7-aminocoumarin-4-methanesulfonic acid (ACMS), a highly water-soluble dye, and their suitability for droplet-based microfluidics applications. Both substrate and product had the required spectral characteristics and remained confined in droplets from hours to days. As a model experiment, a phenylacetylated ACMS was synthesized and used as a fluorogenic substrate of Escherichia coli penicillin G acylase. Kinetic parameters (k(cat) and K(M)) measured in bulk and in droplets on-chip were very similar, demonstrating the suitability of this synthesis strategy to produce a variety of ACMS-based substrates for assaying amidase activities both in microtiter plate and droplet-based microfluidic formats.