A digital microfluidic method for multiplexed cell-based apoptosis assays

Abstract

Digital microfluidics (DMF), a fluid-handling technique in which picolitre-microlitre droplets are manipulated electrostatically on an array of electrodes, has recently become popular for applications in chemistry and biology. DMF devices are reconfigurable, have no moving parts, and are compatible with conventional high-throughput screening infrastructure (e.g., multiwell plate readers). For these and other reasons, digital microfluidics has been touted as being a potentially useful new tool for applications in multiplexed screening. Here, we introduce the first digital microfluidic platform used to implement parallel-scale cell-based assays. A fluorogenic apoptosis assay for caspase-3 activity was chosen as a model system because of the popularity of apoptosis as a target for anti-cancer drug discovery research. Dose-response profiles of caspase-3 activity as a function of staurosporine concentration were generated using both the digital microfluidic method and conventional techniques (i.e., pipetting, aspiration, and 96-well plates.) As expected, the digital microfluidic method had a 33-fold reduction in reagent consumption relative to the conventional technique. Although both types of methods used the same detector (a benchtop multiwell plate reader), the data generated by the digital microfluidic method had lower detection limits and greater dynamic range because apoptotic cells were much less likely to de-laminate when exposed to droplet manipulation by DMF relative to pipetting/aspiration in multiwell plates. We propose that the techniques described here represent an important milestone in the development of digital microfluidics as a useful tool for parallel cell-based screening and other applications.

Microgels on-demand

“…At 4 h after seeding in microgels, viability was B100%; while at 24 h after seeding, viability was B80%. The effects of electrostatic actuation on cell health were not explicitly evaluated here, but previous studies with 2D adherent or suspension cell culture have reported no or negligible effects on cell viability/morphology 24,45 or gene expression 46 when compared with non-actuated cells (as the electrical field drops across the insulating layer rather than droplets containing cells). As shown in Fig.…”

“…We hypothesized that DMF, which has been previously reported to be useful for gentle handling of weakly adhered apoptotic cells 24 , might also be useful for non-disruptive reagent delivery to cell constructs grown in 3D microgels. The results in .…”

“…This technology facilitates electrostatic manipulation of discrete nano-and micro-litre droplets across open-electrode arrays providing the advantages of single-sample addressability, automation and parallelization. Variations of this platform have been demonstrated for a broad range of applications, including proteomics 20,21 , cell culture and analysis [22][23][24] , immunoassays 25,26 , chemical synthesis 27,28 and on-chip lasing 29 . Recently, the unique geometry of DMF has been exploited for handling and addressing of 3D solids, such as paper discs for genetic screening 30,31 , polymer monoliths for sample extractions 32 and agarose discs for scaffolding applications 20,33 .…”

Microfluidics for cell-based high throughput screening platforms—A review

“…The key mechanisms is that the droplet contact angle depends on the electric field applied between the droplet and the chip surface. Recently, DMF has been applied in the study of cell apoptosis as a function of staurosporine concentration, achieving a 33-fold reduction in reagent consumption compared with conventional methods [65]. Another advantage of DMF is that it is compatible with conventional high-throughput screening instruments, but it can result in lower detection limits and larger dynamic range because apoptotic cells are much less likely to delaminate when exposed to droplet manipulation by DMF relative to pipetting/aspiration in multiwell plates.…”

Hydrogel discs for digital microfluidics

“…38 DMF enables facile control over many different reagents for multi-step processes, a property that has been useful for enzyme assays 27,[39][40][41] and for applications involving cells. [42][43][44][45][46][47][48][49][50][51] In these initial applications, enzymatic bioreactors and cell culture and assays were implemented in homogeneous aqueous droplets manipulated by DMF. We propose that the marriage of DMF with hydrogels represents a welcome combination for forming miniaturized enzyme bioreactors and 3D cell culture scaffolds.…”