Virtual microwells for digital microfluidic reagent dispensing and cell culture

Abstract

Digital microfluidic (DMF) liquid handling includes active (electrostatic) and passive (surface tension) mechanisms for reagent dispensing. Here we implement a simple and straightforward Teflon-AF liftoff protocol for patterning hydrophilic sites on a two-plate device for precise passive dispensing of reagents forming virtual microwells--an analogy to the wells found on a microtitre plate. We demonstrate here that devices formed using these methods are capable of reproducible dispensing of volumes ranging from ~80 to ~800 nL, with CVs of 0.7% to 13.8% CV. We demonstrate that passive dispensing is compatible with DMF operation in both air and oil, and provides for improved control of dispensed nano- and micro- litre volumes when compared to active electrostatic dispensing. Further, the technique is advantageous for cell culture and we report the first example of reagent dispensing on a single-plate DMF device. We anticipate this method will be useful for a wide range of applications--particularly those involving adherent cell culture and analysis.

Microgels on-demand

“…2). This technique has been used previously for non-gelling fluids (known in DMF as 'passive dispensing' 22 and in non-DMF methods as 'surface energy traps' 35 ), but this is the first application to exploit this technique for hydrogels. Sub-droplets of sol-phase hydrogel materials deposited onto hydrophilic sites can be cross-linked to form pillar-shaped microgels.…”

“…Each window straddled the interface between two actuation electrodes. DMF device top plates bearing hydrophilic sites were formed by performing a Teflon liftoff procedure on ITO-coated glass substrates as detailed previously 22 . The sites were 0.5-2 mm in diameter and were formed in arrays of 5, 8, 16 or 32 per substrate.…”

“…DMF device fabrication. DMF devices were fabricated using standard photolithography and metal etching, as detailed previously 22 . The bottom-plate device design featured an array of 2.2 Â 2.2 mm chromium actuation electrodes and in certain cases included an array of 1-mm-diameter optical windows (that is, circular regions free from chromium) with 9 mm between each window.…”

“…An open-source, automated DMF actuation system called 'DropBot' (described in detail elsewhere 55 ) was used to program and manage the application of driving potentials of 120-140 V RMS generated by amplifying the sine wave output of a built-in function generator operating at 10 kHz. Reagents were loaded and dispensed, moved and merged as described previously 22 . In all DMF experiments, reagent solutions were supplemented with 0.02% Pluronics F68 except for sol-state Geltrex and collagen that were supplemented with 0.02% Pluronics F127 (ref.…”

“…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 .…”