Virtual microwells for digital microfluidic reagent dispensing and cell culture

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 similar to 80 to similar 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.