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 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. Virtual microwells are formed by passive dispensing on a digital microfluidic platform improving reagent dispensing and cell culture on one- and two-plate devices.