Ashleigh B. Theberge

Microdroplets in Microfluidics: An Evolving Platform for Discoveries in Chemistry and Biology

Microdroplets in microfluidics offer a great number of opportunities in chemical and biological research. They provide a compartment in which species or reactions can be isolated, they are monodisperse and therefore suitable for quantitative studies, they offer the possibility to work with extremely small volumes, single cells, or single molecules, and are suitable for high-throughput experiments. The aim of this Review is to show the importance of these features in enabling new experiments in biology and chemistry. The recent advances in device fabrication are highlighted as are the remaining technological challenges. Examples are presented to show how compartmentalization, monodispersity, single-molecule sensitivity, and high throughput have been exploited in experiments that would have been extremely difficult outside the microfluidics platform.

Controlling the Retention of Small Molecules in Emulsion Microdroplets for Use in Cell-Based Assays

Water-in-oil microdroplets in microfluidics are well-defined individual picoliter reaction compartments and, as such, have great potential for quantitative high-throughput biological screening. This, however, depends upon contents of the droplets not leaking out into the oil phase. To assess the mechanism of possible leaking, the retention of various fluorescein derivatives from droplets formed in mineral oil and stored for hours in a reservoir on chip was studied. Leaking into the oil phase was observed and was shown to be dependent on the nature of the compounds and on the concentration of the silicone-based polymeric surfactant Abil EM 90 used. In experiments in which droplets filled with fluorescein were mixed with droplets filled with only buffer, the rate of efflux from filled droplets to empty droplets was dependent on the number of neighboring droplets of different composition. Buffer droplets with five fluorescein-containing neighbors took up the fluorophore 4.5 times faster than buffer droplets without fluorescein neighbors. The addition of bovine serum albumin (BSA) substantially reduced leaking. A formulation with 5% BSA reduces leaking of the fluorophore from 45% to 3%. Inclusion of BSA enabled experiments to be carried out over periods up to 18 h, without substantial leaking (<5%). We demonstrate the utility of this additive by following the enzymatic activity of alkaline phosphatase expressed by Escherichia coli cells. The ability to reliably compartmentalize genotype (cell) and phenotype (reaction product) is the basis for using microdroplets in directed evolution studies, and the approaches described herein provide a test system for assessing emulsion formulations for such purposes.

Suspended microfluidics

Although the field of microfluidics has made significant progress in bringing new tools to address biological questions, the accessibility and adoption of microfluidics within the life sciences are still limited. Open microfluidic systems have the potential to lower the barriers to adoption, but the absence of robust design rules has hindered their use. Here, we present an open microfluidic platform, suspended microfluidics, that uses surface tension to fill and maintain a fluid in microscale structures devoid of a ceiling and floor. We developed a simple and ubiquitous model predicting fluid flow in suspended microfluidic systems and show that it encompasses many known capillary phenomena. Suspended microfluidics was used to create arrays of collagen membranes, mico Dots (μDots), in a horizontal plane separating two fluidic chambers, demonstrating a transwell platform able to discern collective or individual cellular invasion. Further, we demonstrated that μDots can also be used as a simple multiplexed 3D cellular growth platform. Using the μDot array, we probed the combined effects of soluble factors and matrix components, finding that laminin mitigates the growth suppression properties of the matrix metalloproteinase inhibitor GM6001. Based on the same fluidic principles, we created a suspended microfluidic metabolite extraction platform using a multilayer biphasic system that leverages the accessibility of open microchannels to retrieve steroids and other metabolites readily from cell culture. Suspended microfluidics brings the high degree of fluidic control and unique functionality of closed microfluidics into the highly accessible and robust platform of open microfluidics.

Suzuki–Miyaura coupling reactions in aqueous microdroplets with catalytically active fluorous interfaces

Using microfluidic techniques and a novel fluorous-tagged palladium catalyst, we generated droplet reactors with catalytically active walls and used these compartments for small molecule synthesis.

Generation of Picoliter Droplets with Defined Contents and Concentration Gradients from the Separation of Chemical Mixtures

There has been an increasing drive toward miniaturizing and accelerating experiments with droplet-based microfluidics across the chemical disciplines. Current applications take advantage of the numerous techniques for manipulating nano- to femtoliter droplets within microfluidic devices. To expand the range of possible applications, we have developed a method for compartmentalizing pure compounds within droplets, at a gradient of concentrations, starting from chemical mixtures. In this technique, a mixture is injected into an ultra performance liquid chromatography (UPLC) system, and droplets are generated from the LC output at a frequency high enough to fraction each compound into approximately 10(5) droplets, compartmentalizing pure compounds into a sequence of droplets with a range of concentrations spanning 2-3 orders of magnitude. Here we used fluorescent dyes to quantify the concentration profile of the droplet collections, and to demonstrate the correspondence between the concentration profile of the droplets and the compound elution profile monitored with a UV absorbance detector, allowing the use of compounds that are not fluorescently labeled but show UV absorbance. Hence this technique is applicable to a wide variety of applications that require both compound purity and the ability to probe a variety of concentrations, such as drug screening and titrations.

Investigation of “On Water” Conditions Using a Biphasic Fluidic Platform

Water is the most abundant liquid on the planet and is a unique solvent owing to the internal structure arising from the hydrogen-bond network. It is known that water has a strong effect on the reactivity of dissolved hydrophobic organic molecules. The rate enhancements in water have been attributed to many factors, including “enforced hydrophobic interactions”, enhanced hydrogen bonding of the transition state, the unusually high “cohesive energy density” of water, and in more general terms to the decrease of the hydrophobic surface area of the reactants during the activation process, combined with hydrogen-bond stabilization of the polarized activated complex. 17] In a landmark paper, Sharpless and co-workers studied a range of reactions accelerated by water despite the non-solubility of the reactants under so-called “on water” conditions. Although a large number of studies have exploited these “on water” conditions and have reported rate enhancements, there is no conclusive agreement on the molecular origin of the effect. One major reason for our limited understanding of “on water” reactions lies in the very nature of the experiment; typically, “on water” reactions are carried out by vigorously stirring two immiscible phases. On a laboratory scale, the water surface area and the surface-tovolume ratio cannot be controlled reproducibly as they are strongly dependent on stirring rates, volumes, ratio of water to organic phase, and the size of both the stirrer bar and the reaction flask. The droplet sizes in the emulsions that are formed during the reaction are unknown, making any estimate of the effect of the water surface on the reaction rate an educated guess. As a result, there have been no systematic studies exploring the “on water” effect and it is still unclear how much the water surface really contributes to the measured reaction rates. The aim of the present study is to quantify the “on water” effect per unit area of interface for the first time. We present a fluidic approach to generate precisely defined organic– water interfaces, which allows us to systematically probe the influence of the water surface on chemical reactions. Monodisperse water and organic phase plugs (typical frequencies around 0.06 Hz) are generated in polytetrafluoroethylene (PTFE) tubing by connecting an aqueous flow with an organic flow using a chlorotrifluoroethylene (CTFE) cross-junction (Figure 1a). 21] This approach gives excellent control over the water surface area by changing the relative flow rates of the water and organic phase or the internal diameter of the tubing (Figure 1 b). Our study focuses on two model reactions: the cycloaddition of quadricyclane (1) and diethyl azodicarboxylate (DEAD; Figure 1c), and the ene reaction between b-pinene (2) and DEAD (Figure 1d). These reactions were shown by Sharpless and co-workers to be significantly accelerated under “on water” conditions. 24] DEAD is shock and light sensitive and commercially available as a 40 wt% solution in toluene; therefore we performed all reactions in toluene and in toluene “on water”, with concentrations in the 1m to 3m range. Furthermore, bulk experiments showed (Supporting Information, Figure S1) that the reaction rates vary strongly during the reaction, and therefore we concentrated our studies on reactions with conversions under 10%, where the percent conversion was linear with time. As shown in Figure 2a, the “on water” reaction between 1 and DEAD was accelerated to 4.3 0.2% and 6 0.2% conversion after 60 min “on water” for water– organic (Qw/Qo) flow ratios of 1:1 (Q1) and 2:1 (Q2), respectively, compared to only 1.1 0.2 % conversion in toluene. This result indicated that the reaction takes place because of the introduction of water. It should be noted that when sodium dodecyl sulfate (SDS) was added to the aqueous phase the reaction between 1 and DEAD essentially stopped, with percent conversions closer to those of the reaction in toluene only (Supporting Information, Figure S2). In contrast, we found that the “on water” effect for the reaction of 2 with DEAD is much smaller (Figure 2 b). However, the reaction was sensitive to changes in the water surface area as demonstrated by the 5 0.3% increase in conversion after doubling the water–organic (Qw/Qo) flow ratio, which resulted in a concomitant increase in the water–organic surface area. We determined the order of the reaction by systematically varying the concentration of each reactant between 0.5m and 3m (Supporting Information, Figure S3, Table S1, and Table S2). The reactions are close to first order in 1, 2, and [*] S. Mellouli, L. Bousekkine, Prof. Dr. W. T. S. Huck Institute for Molecules and Materials, Radboud University Nijmegen Heyendaalseweg 135, 6525 AJ Nijmegen (The Netherlands) E-mail: w.huck@science.ru.nl

Microbial metabolomics in open microscale platforms

The microbial secondary metabolome encompasses great synthetic diversity, empowering microbes to tune their chemical responses to changing microenvironments. Traditional metabolomics methods are ill-equipped to probe a wide variety of environments or environmental dynamics. Here we introduce a class of microscale culture platforms to analyse chemical diversity of fungal and bacterial secondary metabolomes. By leveraging stable biphasic interfaces to integrate microculture with small molecule isolation via liquid–liquid extraction, we enable metabolomics-scale analysis using mass spectrometry. This platform facilitates exploration of culture microenvironments (including rare media typically inaccessible using established methods), unusual organic solvents for metabolite isolation and microbial mutants. Utilizing Aspergillus, a fungal genus known for its rich secondary metabolism, we characterize the effects of culture geometry and growth matrix on secondary metabolism, highlighting the potential use of microscale systems to unlock unknown or cryptic secondary metabolites for natural products discovery. Finally, we demonstrate the potential for this class of microfluidic systems to study interkingdom communication between fungi and bacteria.

Microfluidic multiculture assay to analyze biomolecular signaling in angiogenesis

Angiogenesis (the formation of blood vessels from existing blood vessels) plays a critical role in many diseases such as cancer, benign tumors, and macular degeneration. There is a need for cell culture methods capable of dissecting the intricate regulation of angiogenesis within the microenvironment of the vasculature. We have developed a microscale cell-based assay that responds to complex pro- and antiangiogenic soluble factors with an in vitro readout for vessel formation. The power of this system over traditional techniques is that we can incorporate the whole milieu of soluble factors produced by cells in situ into one biological readout (vessel formation), even if the identity of the factors is unknown. We have currently incorporated macrophages, endothelial cells, and fibroblasts into the assay, with the potential to include additional cell types in the future. Importantly, the microfluidic platform is simple to operate and multiplex to test drugs targeting angiogenesis in a more physiologically relevant context. As a proof of concept, we tested the effect of an enzyme inhibitor (targeting matrix metalloproteinase 12) on vessel formation; the triculture microfluidic assay enabled us to capture a dose-dependent effect entirely missed in a simplified coculture assay (p < 0.0001). This result underscores the importance of cell-based assays that capture chemical cross-talk occurring between cell types. The microscale dimensions significantly reduce cell consumption compared to conventional well plate platforms, enabling the use of limited primary cells from patients in future investigations and offering the potential to screen therapeutic approaches for individual patients in vitro.

Effect of Microculture on Cell Metabolism and Biochemistry: Do Cells Get Stressed in Microchannels?

Microfluidics is emerging as a promising platform for cell culture, enabling increased microenvironment control and potential for integrated analysis compared to conventional macroculture systems such as well plates and Petri dishes. To advance the use of microfluidic devices for cell culture, it is necessary to better understand how miniaturization affects cell behavior. In particular, microfluidic devices have significantly higher surface-area-to-volume ratios than conventional platforms, resulting in lower volumes of media per cell, which can lead to cell stress. We investigated cell stress under a variety of culture conditions using three cell lines: parental HEK (human embryonic kidney) cells and transfected HEK cells that stably express wild-type (WT) and mutant (G601S) human ether-a-go-go related gene (hERG) potassium channel protein. These three cell lines provide a unique model system through which to study cell-type-specific responses in microculture because mutant hERG is known to be sensitive to environmental conditions, making its expression a particularly sensitive readout through which to compare macro- and microculture. While expression of WT-hERG was similar in microchannel and well culture, the expression of mutant G601S-hERG was reduced in microchannels. Expression of the endoplasmic reticulum (ER) stress marker immunoglobulin binding protein (BiP) was upregulated in all three cell lines in microculture. Using BiP expression, glucose consumption, and lactate accumulation as readouts we developed methods for reducing ER stress including properly increasing the frequency of media replacement, reducing cell seeding density, and adjusting the serum concentration and buffering capacity of culture medium. Indeed, increasing the buffering capacity of culture medium or frequency of media replacement partially restored the expression of the G601S-hERG in microculture. This work illuminates how biochemical properties of cells differ in macro- and microculture and suggests strategies that can be used to modify cell culture protocols for future studies involving miniaturized culture platforms.

Design of a novel class of biphenyl CETP inhibitors

A new class of CETP inhibitors was designed and prepared. These compounds are potent both in vitro and in vivo. The most active compound (12d) has shown an ability to raise HDL significantly in transgenic mouse PD model.