Multilayered Digital Microfluidic Chip for Cell‐Based Assays

ABSTRACT Most conventional digital microfluidic (DMF) chips face challenges in balancing low actuation voltage, dielectric robustness, and minimal current leakage, thereby limiting their effectiveness and use for biological applications. In this work, we present a multilayered digital microfluidic (mDMF) chip incorporating a multilayer dielectric stack of silicon nitride and parylene C to achieve an optimal balance of these properties. The multilayered and compact electrode design creates two‐fold more operational space, while the multilayer dielectric structure reduces actuation voltages down to 50 V, which demotes a stable two‐fold reduction to conventional standards. Finite element analysis was employed to optimize the dielectric thicknesses, ensuring minimal current leakage under 10 nA, which is 100‐fold below the biological detection threshold. Experimental validation was conducted through droplet actuation and water contact angle characterizations, identifying the optimal dielectric composition as 400‐nm thick silicon nitride and 1‐µm thick parylene C layer. The utility of mDMF chips in biological assays was demonstrated by culturing human‐derived macrophages and fibroblasts under varying concentrations of cytochalasin D. Results indicated comparable experimental performance to conventional well plate assays, while utilizing over 2 orders of magnitude less sample volume. This advancement highlights the efficiency of DMF for cell‐based assays and facilitates wider biomedical applications.