DIB-BOT: An open-source hardware approach for high throughput droplet interface bilayer deposition

Droplet interface bilayers (DIBs) provide a controlled lipid environment for the single-molecule investigation of a range of biologically relevant membrane-bound processes and have garnered attention for their potential applications in bottom-up artificial cells, biosensing, and biophysics. However, the fabrication of DIBs is currently hindered by time-consuming processes and specialized equipment. These fabrication limitations prevent the scale-up of DIB assays, making it difficult to generate the large data sets required to achieve statistically significant conclusions in single-molecule biological assays where heterogeneous behaviour is often observed. This research describes an open-source solution, dubbed "DIB-BOT," constructed by coupling a nanoinjector with an entry-level 3D printer. We present DIB-BOT as a platform to achieve rapid, reproducible, and reliable fabrication of large numbers of DIBs, addressing the limitations of manual methods. Leveraging commercially available off-the-shelf components, DIB-BOT exhibits high spatial reproducibility, minimal user input, and the ability to scale experiments rapidly. Here we demonstrate the utility of the system by integrating pairwise droplet assembly with a fluorescence plate-reader to execute a biologically relevant assay. When compared with manual DIB fabrication, the DIB-BOT had a tenfold reduction in droplet volume error, a threefold reduction in positional error, and 100% droplet yield. Overall, this method has potential to reduce entry barriers to the use of DIB methods, broadening the applications of DIB research, and generating higher quality data sets.