Abstract
Traditionally, the use of DNA origami nanostructures (DONs) to study early cell signaling processes has been conducted using standard laboratory equipment with DONs typically utilized in solution. Surface-based technologies simplify the microscopic analysis of cells treated with DON agents by anchoring them to solid substrates, thus avoiding the complications of receptor-mediated endocytosis. A robust microfluidic platform for real-time monitoring and precise functionalization of surfaces with DONs was developed here. The combination of controlled flow conditions with an upright total internal reflection fluorescence microscope enabled the kinetic analysis of the immobilization of DONs on DNA-functionalized surfaces. The results revealed that DON morphology and binding tags influence the binding kinetics and that DON hybridization on surfaces is more effective in microfluidic devices with larger-than-standard dimensions, addressing the low diffusivity challenge of DONs. The platform enabled the decoration of DONs with protein-binding ligands and in situ investigation of ligand occupancy on DONs to produce high-quality bioactive surfaces. These surfaces were used to recruit and activate the epidermal growth factor receptor (EGFR) through clustering in the membranes of living cancer cells (MCF-7) using an antagonistic antibody (Panitumumab). The activation was quantified depending on the interligand distances of the EGFR-targeting antibody.