Gravity-induced tunable asymmetric droplet splitting for flexible and precise reagent formulation on vertical digital microfluidic devices

electrowetting on dielectric (EWOD) force. However, droplet splitting behavior in conventional digital microfluidic devices is limited by the geometry of actuating electrodes. In this study, we proposed a gravity-induced size-tuning splitting (GITS) method, which has no requirements for specialized electrodes or complicated chip configurations. Both experimental and simulation results demonstrated that gravity facilitates the droplet generation by directionally enhancing the EWOD force in a vertical digital microfluidic chip. Further observation revealed that the size tunability was affected by the droplet volume, voltage amplitude, and especially contact line ratios between droplet and electrodes. Moreover, to achieve reliable on-chip operations, the critical size of the droplet for passive dropping was investigated, which exhibited a functional relationship with the gap height. Then GITS was implemented by integrating an artificial intelligence (AI)-driven feedback control of the contact line and the gravity induced droplet dropping. As a result, it achieved wide splitting ratios from 1 to 7.33, with the coefficient of variation below 3%. Finally, GITS was applied to manage reagents of various sizes for on-chip cell viability assays, demonstrating its potential for flexible reagent configuration in future biomedical applications.