A Scalable Droplet Interface Bilayer Assembly Platform For Ensemble And Single Channel Biophysics

The transport of DNA and RNA through ion channels in both biological and technological systems has been studied with increasing intensity over the past two decades by biologists, chemists and biophysicists. The prevailing experimental method by which to investigate ion channels at single molecule resolution makes use of the planar lipid bilayer (PLB) system. In this synthetic system, ion channels are reconstituted in artificial lipid bilayers. While the power of this technique has allowed the biophysical characterization of a large number of ion channels, its technical and experimental limitations are well-documented.;With respect to these limitations we have made two advances. The first advance, the wicking method, is an improvement upon the PLB system. This new method has two central features. First, it reduces system complexity and removes lamellarity ambiguity by means of an empirically derived capacitance threshold. Second, it reduces the long reconstitution times associated with passive fusion of proteoliposomes to PLBs. Taken together, these features enable an increase in throughput by up to two orders of magnitude.;In spite of the improvement made by the wicking method, the PLB system is still fraught with the remaining problems of instability, restricted throughput, and requirement of a skilled technical operator. The second technical advance we have made involves a complete departure from the PLB system in favor of the development of a new technological platform based on the droplet interface bilayer (DIB) system. This platform has two central technological features. The first is completely clean room-independent fabrication of droplet interface bilayer scaffolds by means of 3D-printing. The second is a fluidic strategy for bilayer assembly that can be implemented macroscopically in a parallelizable manner. The coupling of these features has enabled the establishment of a rapidly prototypable, fully configurable and scalable platform that allows rapid synthetic reconstitution of ion channels. The platform has been validated using the biological nanopores gramicidin A and S. aureus alpha hemolysin. Ensemble and single channel electrophysiology of these nanopores highlights the platform's robustness with respect to stability and dynamic range, features not achievable in the PLB system.