Microarrays of phospholipid bilayers generated by inkjet printing

We report an efficient and reproducible method to generate a microarray of model biological membranes on a solid substrate by applying the inkjet printing technology. Although inkjet printing is currently widely used for industrial fabrication processes, including biological materials, printing lipid membranes remains technically challenging due to the hydrophobic nature of droplets and instability of the lipid bilayer structure against dehydration. In the present study, we printed lipids onto a glass substrate covered with a micropatterned membrane of a polymeric phospholipid bilayer. Polymeric bilayers were formed by the lithographic photopolymerization of a diacetylene-containing phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC). After removal of nonpolymerized DiynePC with a detergent solution, natural lipid membranes were incorporated into the polymer-free regions (corrals) by using an electric-field-based inkjet printing device that can eject subfemtoliter volume droplets. To avoid rapid dehydration and destabilization, we preprinted an aqueous solution containing agarose and trehalose onto the corrals and subsequently printed lipid suspensions ("two-step-printing method"). After rinsing, stable lipid bilayer membranes were formed in the corrals. The bilayers were continuous and fluid as confirmed by fluorescence recovery after photobleaching. We could introduce multiple bilayer patches having different lipid compositions into the neighboring corrals. The present results demonstrate that the combination of a patterned polymeric bilayer and inkjet printing technology enables efficient, reliable, and scalable generation of the model membrane microarrays having varied compositions.