A programmable and automated optical electrowetting-on-dielectric (oEWOD) driven platform for massively parallel and sequential processing of single cell assay operations.

Recently, there has been an increasing emphasis on single cell profiling for high-throughput screening workflows in drug discovery and life sciences research. However, the biology underpinning these screens is often complex and is insufficiently addressed by singleplex assay screens. Traditional single cell screening technologies have created powerful sets of 'omic data that allow users to bioinformatically infer biological function, but have as of yet not empowered direct functional analysis at the level of each individual cell. Consequently, screening campaigns often require multiple secondary screens leading to laborious, time-consuming and expensive workflows in which attrition points may not be queried until late in the process. We describe a platform that harnesses droplet microfluidics and optical electrowetting-on-dielectric (oEWOD) to perform highly-controlled sequential and multiplexed single cell assays in massively parallelised workflows to enable complex cell profiling during screening. Soluble reagents or objects, such as cells or assay beads, are encapsulated into droplets of media in fluorous oil and are actively filtered based on size and optical features ensuring only desirable droplets (e.g. single cell droplets) are retained for analysis, thereby overcoming the Poisson probability distribution. Droplets are stored in an array on a temperature-controlled chip and the history of individual droplets is logged from the point of filter until completion of the workflow. On chip, droplets are subject to an automated and flexible suite of operations including the merging of sample droplets and the fluorescent acquisition of assay readouts to enable complex sequential assay workflows. To demonstrate the broad utility of the platform, we present examples of single-cell functional workflows for various applications such as antibody discovery, infectious disease, and cell and gene therapy.

Microfluidic Technique for the Simultaneous Quantification of Emulsion Instabilities and Lipid Digestion Kinetics.

Quantifying the impact of environmental physicochemical changes on the microstructure of lipid delivery systems is challenging. Therefore, we have developed a methodology to quantify the coalescence of oil-in-water emulsion droplets during lipid digestion in situ on a single droplet level. This technique involves a custom-made glass microfluidic platform, in which oil droplets can be trapped as single droplets, or several droplets per trap. The physicochemical environment can be controlled, and droplet digestion, as well as coalescence, can be visualized. We show that the exchange of the physicochemical conditions in the entire reaction chamber can be reached in under 30 s. Microparticle image velocimetry allowed mapping of the flow profile and demonstrated the tuneability of the shear profile in the device. The extraction of quantitative information regarding the physical characteristics of the droplets during digestion was performed using an automated image analysis throughout the digestion process. Therefore, we were able to show that oil-in-water emulsions stabilized by proteins coalesced under human gastric conditions. This coalescence delayed the overall lipid digestion kinetics. The droplets that coalesced during digestion were hydrolyzed 1.4 times slower than individually trapped droplets. Thus, the microstructural evolution of lipid delivery systems is a crucial factor in lipid digestion kinetics. This novel technique allows the simultaneous quantification of the impact that the physicochemical environment has on both the lipid droplet microstructure and the lipid release patterns.

A Microfluidic Platform for the Rapid Determination of Distribution Coefficients by Gravity-Assisted Droplet-Based Liquid-Liquid Extraction.

The determination of pharmacokinetic properties of drugs, such as the distribution coefficient (D) is a crucial measurement in pharmaceutical research. Surprisingly, the conventional (gold standard) technique used for D measurements, the shake-flask method, is antiquated and unsuitable for the testing of valuable and scarce drug candidates. Herein, we present a simple microfluidic platform for the determination of distribution coefficients using droplet-based liquid-liquid extraction. For simplicity, this platform makes use of gravity to enable phase separation for analysis and is 48 times faster and uses 99% less reagents than performing an equivalent measurement using the shake-flask method. Furthermore, the D measurements achieved in our platform are in good agreement with literature values measured using traditional shake-flask techniques. Since D is affected by volume ratios, we use the apparent acid dissociation constant, pK', as a proxy for intersystem comparison. Our platform determines a pK' value of 7.24 ± 0.15, compared to 7.25 ± 0.58 for the shake-flask method in our hands and 7.21 for the shake-flask method in the literature. Devices are fabricated using injection molding, the batchwise fabrication time is <2 min per device (at a cost of $1 U.S. per device), and the interdevice reproducibility is high.

Droplet confinement and leakage: Causes, underlying effects, and amelioration strategies.

The applicability of droplet-based microfluidic systems to many research fields stems from the fact that droplets are generally considered individual and self-contained reaction vessels. This study demonstrates that, more often than not, the integrity of droplets is not complete, and depends on a range of factors including surfactant type and concentration, the micro-channel surface, droplet storage conditions, and the flow rates used to form and process droplets. Herein, a model microfluidic device is used for droplet generation and storage to allow the comparative study of forty-four different oil/surfactant conditions. Assessment of droplet stability under these conditions suggests a diversity of different droplet failure modes. These failure modes have been classified into families depending on the underlying effect, with both numerical and qualitative models being used to describe the causative effect and to provide practical solutions for droplet failure amelioration in microfluidic systems.

A 3D-printed microcapillary assembly for facile double emulsion generation.

The design, fabrication and testing of facile microcapillary device assembly, suitable for monodisperse double emulsion production is reported. The interface is fabricated in a direct and rapid manner via 3D printing and shown to be robust in the controllable generation of both single and double emulsions at high generation frequencies.

The past, present and potential for microfluidic reactor technology in chemical synthesis.

The past two decades have seen far-reaching progress in the development of microfluidic systems for use in the chemical and biological sciences. Here we assess the utility of microfluidic reactor technology as a tool in chemical synthesis in both academic research and industrial applications. We highlight the successes and failures of past research in the field and provide a catalogue of chemistries performed in a microfluidic reactor. We then assess the current roadblocks hindering the widespread use of microfluidic reactors from the perspectives of both synthetic chemistry and industrial application. Finally, we set out seven challenges that we hope will inspire future research in this field.

Droplet-based microfluidic platform for high-throughput, multi-parameter screening of photosensitizer activity.

We present a fully integrated droplet-based microfluidic platform for the high-throughput assessment of photodynamic therapy photosensitizer (PDT) efficacy on Escherichia coli. The described platform is able to controllably encapsulate cells and photosensitizer within pL-volume droplets, incubate the droplets over the course of several days, add predetermined concentrations of viability assay agents, expose droplets to varying doses of electromagnetic radiation, and detect both live and dead cells online to score cell viability. The viability of cells after encapsulation and incubation is assessed in a direct fashion, and the viability scoring method is compared to model live/dead systems for calibration. Final results are validated against conventional colony forming unit assays. In addition, we show that the platform can be used to perform concurrent measurements of light and dark toxicity of the PDT agents and that the platform allows simultaneous measurement of experimental parameters that include dark toxicity, photosensitizer concentration, light dose, and oxygenation levels for the development and testing of PDT agents.

Continuous and segmented flow microfluidics: applications in high-throughput chemistry and biology.

This account highlights some of our recent activities focused on developing microfluidic technologies for application in high-throughput and high-information content chemical and biological analysis. Specifically, we discuss the use of continuous and segmented flow microfluidics for artificial membrane formation, the analysis of single cells and organisms, nanomaterial synthesis and DNA amplification via the polymerase chain reaction. In addition, we report on recent developments in small-volume detection technology that allow access to the vast amounts of chemical and biological information afforded by microfluidic systems.

The naphyrone story: The alpha or beta-naphthyl isomer?

Naphyrone (naphthylpyrovalerone, O-2482) has been recently advertised for purchase on a number of websites. This compound has been viewed as a so-called 'legal high' and was classified as a controlled drug under the UK Misuse of Drugs Act 1971 in mid-July 2010. So far, naphyrone is commonly equated with 1-naphthalen-2-yl-2-pyrrolidin-1-yl-pentan-1-one (ß-naphyrone) but analytical characterization of two naphyrone samples revealed the existence of a novel isomer consistent with 1-naphthalen-1-yl-2-pyrrolidin-1-yl-pentan-1-one (α-naphyrone). Analyses of both α- and ß-naphyrone were carried out using gas chromatography ion trap (EI/CI) mass spectrometry and 1D/2D nuclear magnetic resonance spectroscopy. This provides the first report of α-naphyrone in the scientific literature and the ability to differentiate it from the ß-isomer should be of interest to forensic and clinical communities.

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.

A one-step protocol for the chemical derivatisation of glass microfluidic devices.

A simple and robust derivatisation system for glass and silica microdevices is described. The device surface is coated in a one-step treatment with a highly cross-linked polystyrene/divinylbenzene/allylsiloxane copolymer. The surface derivatisation is highly resistant to solvents, acids, bases and oxidising or reducing agents.

A method for rapid reaction optimisation in continuous-flow microfluidic reactors using online Raman spectroscopic detection.

An extremely rapid tool for continuous flow synthetic process optimisation is described. A microfluidic reaction system operating in continuous flow is used in conjunction with confocal Raman microscopy to afford rapid molecule synthesis and product quantitation. Accordingly, the approach allows for rapid reaction optimisation within a continuous flow system. Specifically, the catalytic oxidation of isopropyl alcohol (IPA) to acetone using tetra-N-propylammonium perruthanate (TPAP)/N-methylmorpholine N-oxide (NMO) in a radial interdigitated micromixer is studied as a model reaction system. The composition of the reaction effluent can be determined with great facility and information relating to catalyst/co-oxidant ratios, catalyst turnovers and reaction endpoints extracted. Specifically, variation of catalyst and co-oxidant volumetric flow rates between 0 and 50 microL min(-1) is used to vary reactant concentrations, define reaction residence times and control product conversions between 0 and 100%. The rapid nature of the system allows chemical information to be gathered and utilised on a sub-minute timescale.

Microfluidic systems for high-throughput and combinatorial chemistry.

Demands on modern combinatorial chemistry have necessitated massive investment in new synthetic technologies that will allow the production of highly pure drug candidates on short timescales. The advent of microfluidic reactor technologies has recently provided a new tool for the combinatorial chemist, offering the promise of ultra-high-throughput solution-phase chemistries. This review describes why such microscale systems provide unique environments for performing high-efficiency molecular synthesis, details some of the most important recent developments in the field and assesses the true applicability of micro-engineered reactors in high-throughput compound synthesis.

Precise temperature control in microfluidic devices using Joule heating of ionic liquids.

Microfluidic devices for spatially localised heating of microchannel environments were designed, fabricated and tested. The devices are simple to implement, do not require complex manufacturing steps and enable intra-channel temperature control to within +/-0.2 degrees C. Ionic liquids held in co-running channels are Joule heated with an a.c. current. The nature of the devices means that the internal temperature can be directly assessed in a facile manner.