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.

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 IC 50 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 IC 50 of 27 0.83 μM.high-throughput screening | HTS | small molecule library I n the early 16th century the Swiss chemist Paracelsus declared "all substances are poisons, there is none which is not a poison; only the right dose makes a substance non-poisonous." This idea that the biological effects of a chemical compound are dependent upon its concentration was quantified by A. V. Hill in 1910 (1). However, despite the fact that compounds can display complex concentration-dependent relationships, varying in potency, efficacy, and steepness of response, usually just a single measurement at a single concentration (approximately 10 μM) is obtained for each compound in the chemical library during a primary drug screen, even when using state-of-the-art robotic microplate-based screening systems. This results in high numbers of false positives and false negatives (2), as well as the inability to identify subtle, complex pharmacology, such as partial agonism or antagonism. Even when dose-response curves are generated during a quantitative primary screen (3) or, more typically, during the follow-up of a single-point screen, the time and cost limitations mean that the curves typically contain only 7-10 data points each. With ≤10 nonduplicated data points, and as many as four adjustable nonlinear parameters (e.g., in the four-parameter Hill function), the results are highly sensitive to the data quality: For example, the presence of a single outlying data point can substantially alter the fit of the data, unless the outliers are identified and removed (4).We have developed a system that uses droplet-based microfluidics to generate high-quality dose-response data during drug screening. Droplet-based microfluidics is itself a new technology for creating and manipulating picoliter-volume aqueous droplets that function as independent microreactors (for a review see ref. 5). As a result of the miniaturization inherent in this approach, our system (Fig. 1) is capable of generating doseresponse curves at materiall...

Filamentous fungi are an extremely important source of industrial enzymes because of their capacity to secrete large quantities of proteins. Currently, functional screening of fungi is associated with low throughput and high costs, which severely limits the discovery of novel enzymatic activities and better production strains. Here, we describe a nanoliter-range droplet-based microfluidic system specially adapted for the high-throughput sceening (HTS) of large filamentous fungi libraries for secreted enzyme activities. The platform allowed (i) compartmentalization of single spores in ~10 nl droplets, (ii) germination and mycelium growth and (iii) high-throughput sorting of fungi based on enzymatic activity. A 104 clone UV-mutated library of Aspergillus niger was screened based on α-amylase activity in just 90 minutes. Active clones were enriched 196-fold after a single round of microfluidic HTS. The platform is a powerful tool for the development of new production strains with low cost, space and time footprint and should bring enormous benefit for improving the viability of biotechnological processes.

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.