3D Droplet Microfluidic Systems for High-Throughput Biological Experimentation.

Herein, we describe the development of a multilayer droplet microfluidic system for creating concentration gradients and generating microdroplets of varying composition for high-throughput biochemical and cell-based screening applications. The 3D droplet-based microfluidic device consists of multiple PDMS layers, which are used to generate logarithmic concentration gradient reagent profiles. Parallel flow focusing structures are used to form picoliter-sized droplets of defined volumes but of varying composition. As proof of concept, we demonstrate rapid enzymatic activity assays and drug cytotoxicity assays on bacteria. The 3D droplet-based microfluidic platform has the potential to allow for high-efficiency and high-throughput analysis, overcoming the structural limitations of single layer microfluidic systems.

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.

Compartmentalization of electrophoretically separated analytes in a multiphase microfluidic platform.

Herein, we describe the monolithic integration of a multiphase microfluidic system to a microcapillary gel electrophoresis (µCGE) architecture for the complete isolation and storage of separated analyte bands. Within this platform, analyte molecules are separated using microchannel gel electrophoresis, and the eluted bands are stored in a sequence of approximately 40-600 encapsulating microdroplets. Importantly, employing such a system allows for total control of droplet size, shape, and composition. This approach is utilized to separate, optically detect, and encapsulate two fluorescent analytes from a composite sample mixture. Further to this, we subsequently investigate the potential of the system to be used as a concentration gradient generator through analysis of the segmented analyte bands and droplet composition.

Combinatorial microfluidic droplet engineering for biomimetic material synthesis.

Although droplet-based systems are used in a wide range of technologies, opportunities for systematically customizing their interface chemistries remain relatively unexplored. This article describes a new microfluidic strategy for rapidly tailoring emulsion droplet compositions and properties. The approach uses a simple platform for screening arrays of droplet-based microfluidic devices and couples this with combinatorial selection of the droplet compositions. Through the application of genetic algorithms over multiple screening rounds, droplets with target properties can be rapidly generated. The potential of this method is demonstrated by creating droplets with enhanced stability, where this is achieved by selecting carrier fluid chemistries that promote titanium dioxide formation at the droplet interfaces. The interface is a mixture of amorphous and crystalline phases, and the resulting composite droplets are biocompatible, supporting in vitro protein expression in their interiors. This general strategy will find widespread application in advancing emulsion properties for use in chemistry, biology, materials, and medicine.

Lab-chip HPLC with integrated droplet-based microfluidics for separation and high frequency compartmentalisation.

We demonstrate the integration of a droplet-based microfluidic device with high performance liquid chromatography (HPLC) in a monolithic format. Sequential operations of separation, compartmentalisation and concentration counter were conducted on a monolithic chip. This describes the use of droplet-based microfluidics for the preservation of chromatographic separations, and its potential application as a high frequency fraction collector.

Recent advances in microfluidic technologies for biochemistry and molecular biologys.

Advances in the fields of proteomics and genomics have necessitated the development of high-throughput screening methods (HTS) for the systematic transformation of large amounts of biological chemical data into an organized database of knowledge. Microfluidic systems are ideally suited for high-throughput biochemical experimentation since they offer high analytical throughput, consume minute quantities of expensive biological reagents, exhibit superior sensitivity and functionality compared to traditional micro-array techniques and can be integrated within complex experimental work flows. A range of basic biochemical and molecular biological operations have been transferred to chip-based microfluidic formats over the last decade, including gene sequencing, emulsion PCR, immunoassays, electrophoresis, cell-based assays, expression cloning and macromolecule blotting. In this review, we highlight some of the recent advances in the application of microfluidics to biochemistry and molecular biology.

Fluorescence detection methods for microfluidic droplet platforms.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints. In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation. Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented. Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.