by Irwin A. Eydelnant and Aaron R. Wheeler
Digital microfluidics (DMF) is helping to revolutionize miniaturisation in biology. With the ability to support cell culture and perform complex multi-step protocols in sub-microlitre volumes, DMF is being eyed as a potential platform for bringing accessible low-cost high-throughput screening to basic and applied research laboratories.
Miniaturisation in biology
Miniaturisation of biology began with the advent of the microwell plate and is driven by the high-throughput demands of drug screening and the systems approach to biology. The adoption of high-throughput cell-based biological screens as standard practice within the academic laboratory is furthering the need for accessible high-throughput systems. Robotic liquid handling though useful for experimental automation is often inaccessible because of high capital costs (e.g., robotics), large volumes of reagent consumption (e.g., drugs, media, cells) and high turnover of consumables (e.g., microwell plates, pipette tips). These obstacles have motivated the development of miniaturised platforms with the capability to manipulate micro-scale samples. Microfluidic technologies, where liquid manipulation is implemented in micron-scale confined volumes, have provided the tools needed for ultra low volume reagent handling. With an abundance of research groups developing biologically oriented microfluidic technologies, the first generation of laboratory protocols implemented in devices with the footprint of a credit card has been realised.
Microfluidic paradigms
Multiple paradigms of microfluidics, including continuous channel microfluidics, droplet-in-channel microfluidics and digital microfluidics, have emerged, each with its own advantages and disadvantages [1]. Continuous channel microfluidics, where reagent flows are confined within micron scale channels, allows for well controlled serial device operations. The laminar flow properties of these systems allow for flow streams to be constrained within a single channel without mixing. These features have been exploited in biological assays for the formation of well-defined diffusion gradients across cell monolayers, single cell capture and analysis (e.g., PCR, fluorescence microscopy), modelling microvasculature and other studies requiring precise control of the chemical (e.g., growth factors, cytokines) and physical (e.g., shear stress, flow rate) cellular microenvironment. With the incorporation of on-device valves, these devices have been scaled into compartmentalised high-throughput systems. However, because of the inherently complex fabrication protocols required to form such systems, reliance on external equipment (e.g., pumps, valve manifolds), and networks of connecting tubing required for reagent transfer, biologists have been slow to adopt such systems into their routine work-flow.
In droplet-in-channel microfluidics, pico- and nano-litre droplets are generated in a two-phase flow in microchannels. This allows for the formation of thousands of independent droplets per second that can be merged, sorted and reacted. With simple fabrication and high-throughput operation (10-100 kHz) these devices are well positioned for screening in biological experiments. Biocompatible surfactants stabilise the emulsions, allowing for encapsulation of live cells for suspension culture or hydrogel materials for adherent culture. The use of fluorinated oils in the continuous phase permits sufficient oxygen transfer to maintain cell viability for multiple days. The throughput of droplet microfluidic systems is unparalleled; however, such systems are not well suited to multi-step long-term applications involving cells because of the challenges in addressing individual droplets (for media exchange, reagent addition, staining). In addition, both continuous and droplet-in-channel methods typically require dedicated specialised microscopy analysis techniques; this limits their flexibility for integration with standard analytical laboratory equipment. Over the past decade, a third paradigm, digital microfluidics, has emerged as a potential solution to these limitations.
What is digital microfluidics?
Digital microfluidics (or DMF) is a liquid handling technology that permits the independent electrostatic manipulation of individual pico- to micro-litre size droplets across arrays of electrodes [2]. The most common DMF format features droplets sandwiched between two plates, and typical operations include droplet dispensing, splitting, merging and mixing [Figure 1]. The bottom plate is patterned with electrodes buried beneath a hydrophobic insulator. The top plate comprises a contiguous electrode coated with a hydrophobic layer. The hydrophobic coatings are critical to reduce friction forces that can impede droplet movement. When voltages are applied to a driving electrode on the bottom plate relative to a counter-electrode on the top plate, a separation of charge occurs across the insulator acting on ions or dipoles within the droplet. The resulting electrostatic force drives droplet translation. Device fabrication follows basic photolithography and metal etching protocols on a range of substrates including glass, silicone, flexible polyimide films, compact discs, and printed circuit boards (PCBs). Currently a range of techniques for rapid prototyping and mass-production of devices are being investigated, with a particular emphasis on multi-layer PCBs.
DMF compatibility with cell culture
Digital microfluidics is a useful platform for the miniaturisation of cell culture and assays. With the capacity to support both adherent and suspension cell culture, several research groups have demonstrated long-term culture and passaging on-device. Suspension cell culture is well suited to DMF as droplets are manipulated across hydrophobic surfaces that are resistant to adhesion. DMF based viability assays have been performed with comparable results relative to 96-well plate assays and 100-fold reductions in reagent volumes [2]. Droplet mixing by translation allows for cell growth, and droplet splitting and merging are useful for dilution and passaging. For adherent cell culture, multiple strategies have been developed for surface functionalisation including protein deposition, plasma etching and fluorocarbon liftoff [Figure 2A]. Each has been demonstrated for the culture of immortalised cell lines, while the latter has been successfully implemented in the culture of more sensitive cell types including primary and embryonic stem cells [Figure 2B], [3].
The introduction of hydrophilic sites to the hydrophobic coating resulted in the discovery of a novel fluidic phenomenon termed |passive dispensing. As droplets are translated across hydrophilic sites, a portion of the droplet is pinned to the site and a sub-droplet is formed. Passive dispensing has been exploited for controlling volumes for cell seeding and subsequent media and reagent exchange [Figure 2A], [4].
The successful culture of multiple cell types suggests minimal electromagnetic effects on DMF cultured cells. This has been supported by computational modelling of potentials across the device, which demonstrated that the majority of the voltage drop occurs in the dielectric layer and a minimal potential is experienced by the droplet. Biofouling of device surfaces remains the most significant challenge in long-term device operation. Protein adsorption to the hydrophobic surfaces results in droplet pinning thereby restricting droplet translation. The addition of low concentrations of biocompatible surfactants (e.g., Pluronics) improves device function, but does not allow for indefinite device operation.
Novel surfactants and fouling-resistant surface coatings are being investigated to address these issues.
Assays and integration
The real benefits of DMF are realised in the automation of multi-step assays and integration of devices within existing laboratory analytical infrastructure. Live-cell apoptosis assays with cell seeding followed by stimulation, washing and staining steps were recently performed on DMF [5]. These assays were performed in microlitre droplet volumes and did not suffer from cell loss during reagent exchange, a common problem for such assays when performed in microwell plates. Multiplexing of these assays on a single device provides for the ability to quickly and efficiently screen a range of conditions [Figure 2C]. The use of fluorescent apoptosis markers allows for direct device integration with a fluorescent plate reader of the type that is common in research laboratories. In other work, primary cells were cultured for multiple days on DMF [3]. These cells were stimulated with cytokines and their functional responses tested in a monocyte adhesion assay. Further, on-device fixing and staining of these cells followed by epifluorescent microscopy demonstrated compatibility with microscopy for the acquisition of high-quality images. These examples represent a small fraction of the assays that have been implemented by DMF, which is proving useful as a sample prep. tool for surface plasmon resonance, mass spectrometry and electrochemical detection.
The future
One of the visions of digital microfluidics for high throughput biological screening is the eventual development of low-cost smart microwell plates to complement or replace automated liquid handling robotics. Ideally these would function as self-contained cell culture and analysis units capable of multiplexed cell based assays. This will require the combination of robust low-cost devices, novel surfaces that are resistant to biofouling and automation hardware to drive droplet translation. With an increasing number of research groups actively pursuing the development of DMF, the achievement of these goals is promising.
References
1. Squires TM. and Quake SR. Microfluidics: Fluid physics at the nanoliter scale. Rev Mod Phys 2005; 77: 9771026.
2. Choi K, Ng AHC, Fobel R, Wheeler AR. Digital microfluidics. Annu Rev Anal Chem 2012; 5: 413-440.
3. Srigunapalan S, Eydelnant IA, Simmons CA and Wheeler AR. A digital microfluidic platform for primary cell culture and analysis. Lab on a Chip 2012; 12: 369.
4. Eydelnant IA, Uddayasankar U, Li B, Liao MW and Wheeler AR. Virtual microwells for digital microfluidic reagent dispensing and cell culture. Lab on a Chip 2012; 12: 750.
5. Bogojevic D, Chamberlain MD, Barbulovic-Nad I and Wheeler AR. A digital microfluidic method for multiplexed cell-based apoptosis assays. Lab on a Chip 2012; 12: 627.
The authors
Irwin A. Eydelnant a,b, and Aaron R. Wheeler a,b,c,*
a Institute for Biomaterials and Biomedical Engineering, University of Toronto, 164 College St., Toronto, ON, M5S 3G9, Canada
b Donnelly Centre for Cellular and Biomolecular Research, 160 College St, Toronto, ON, M5S 3E1, Canada
c Department of Chemistry, University of Toronto, 80 St. George St., Toronto, ON, M5S 3H6 Canada
* e-mail: aaron.wheeler@utoronto.ca; Tel: +1 (416) 946 3864
