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    Stable trapping of multiple proteins at physiological conditions using nanoscale chambers with macromolecular gates

    Year: 2023

    Authors: Svirelis, Justas; Adali, Zeynep; Emilsson, Gustav; Medin, Jesper; Andersson, John; Vattikunta, Radhika; Hulander, Mats; Järlebark, Julia; Kolman, Krzysztof; Olsson, Oliver; Sakiyama, Yusuke; Lim, Roderick Y. H.; Dahlin, Andreas

    Keywords: Nanopores; Nanoscale biophysics; Nanostructures; Polymer chemistry

    The possibility to detect and analyze single or few biological molecules is very important for understanding interactions and reaction mechanisms. Ideally, the molecules should be confined to a nanoscale volume so that the observation time by optical methods can be extended. However, it has proven difficult to develop reliable, non-invasive trapping techniques for biomolecules under physiological conditions. Here we present a platform for long-term tether-free (solution phase) trapping of proteins without exposing them to any field gradient forces. We show that a responsive polymer brush can make solid state nanopores switch between a fully open and a fully closed state with respect to proteins, while always allowing the passage of solvent, ions and small molecules. This makes it possible to trap a very high number of proteins (500-1000) inside nanoscale chambers as small as one attoliter, reaching concentrations up to 60 gL−1. Our method is fully compatible with parallelization by imaging arrays of nanochambers. Additionally, we show that enzymatic cascade reactions can be performed with multiple native enzymes under full nanoscale confinement and steady supply of reactants. This platform will greatly extend the possibilities to optically analyze interactions involving multiple proteins, such as the dynamics of oligomerization events.
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