On-chip isotachophoresis separation of functional DNA origami capture nanoarrays from cell lysate

Scaffolded DNA origami, a versatile method to construct high yield self-assembled DNA nanostructures, has been investigated to develop water-soluble nanoarrays for label free RNA detection, drug delivery, molecular positioning and recognition, and spatially ordered catalysis of single molecule chemical reactions. Its attributes that facilitate these applications suggest DNA origami as a candidate platform for intracellular targeting. After the interaction with targeted proteins in cell lysate, it is critical to separate and concentrate DNA origami nanoarrays from the crude cell lysate for further analysis. The recent development of microchip isotachophoresis (ITP) provides an alternative robust sample preconcentration and electrophoretic separation method. In this study, we present online ITP for stacking, separation and identification of aptamer-functionalized DNA origami and its thrombin complex in a simple cross-channel fused silica microfluidic chip. In particular, the method achieved separation of a binding complex in less than 5 min and 150-fold signal enhancement. We successfully separated and analyzed the thrombin bound origami-aptamer spiked into cell lysate using on-chip ITP. Our results demonstrate that origami/thrombin nanostructures can be effectively separated from cell lysate using this method and that the structural integrity of the concentrated binding complex is maintained as confirmed by atomic force microscopy (AFM). An ITP-based separation module can be easily coupled to other microchip pre- and post-processing steps to provide an integrated proteomics analysis platform for diagnostic applications.