Abstract
Nanopore technology is widely used for sequencing DNA, RNA, and peptides with single-molecule resolution, for fingerprinting single proteins, and for detecting metabolites. However, the molecular driving forces controlling the analyte capture, its residence time, and its escape have remained incompletely understood. The recently developed Nanopore Electro-Osmotic trap (NEOtrap) is well fit to study these basic physical processes in nanopore sensing, as it reveals previously missed events. Here, we use the NEOtrap to quantitate the electro-osmotic and electrophoretic forces that act on proteins inside the nanopore. We establish a physical model to describe the capture and escape processes, including the trapping energy potential. We verified the model with experimental data on CRISPR dCas9-RNA-DNA complexes, where we systematically screened crucial modeling parameters such as the size and net charge of the complex. Tuning the balance between electrophoretic and electro-osmotic forces in this way, we compare the trends in the kinetic parameters with our theoretical models. The result is a comprehensive picture of the major physical processes in nanopore trapping, which helps to guide the experiment design and signal interpretation in nanopore experiments.