The possibility to detect and analyze single or few biological molecules has proven very important for understanding their function and interaction mechanisms. Ideally, the molecules should be confined to a nanoscale volume so that the observation time can be extended. However, it has proven difficult to develop reliable, non-invasive trapping techniques for biomolecules under physiological conditions. Here we present a new concept for long-term and tether-free 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 (up to ~1 kg/mol). This makes it possible to trap a very high number of proteins (500-1000) inside nanoscale chambers with a volume as small as one attoliter, reaching concentrations up to 60 g/L, which is orders of magnitude higher than for existing container-based technologies. Additionally, our method is fully compatible with parallelization by imaging arrays of nanochambers. As an application example, 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 of single/few molecule analysis by optical methods, and should be particularly useful for studying protein-protein interactions, such as the dynamics of oligomerization.