Single molecules based techniques provide the ultimate toolkit to study complex biological systems. In single-molecule approaches, molecules do not need to be synchronised as in ensemble studies, and rare and/or transient species along a reaction pathway as well as heterogeneity and disorder in a sample can be revealed.Observing and manipulating single molecules, however, is generally complicated, time consuming and withstand several technical limitations that hamper the study of single enzymes to a few selected examples.Here I am proposing to develop a new technology to study single native enzymes that is sensitive, simple and inexpensive, and has a temporal resolution ranging from few ¼seconds to hours.Nanopores have been used to detect single molecules and to investigate mechanisms of chemical reactions at the single molecule level. The basic concept of nanopore analysis is to observe, under an applied potential, the disruption of the flow of ions through the pore caused by the interaction of the molecules of interest with a binding site within the pore.Similarly, small enzymes or functional nucleic acids will be attached to the vestibule of a biological nanopore via disulfide bridge or click chemistry. The conformational changes associated with catalysis will then be observed by the altered ionic flow through the pore. In addition, when a charged chaotropic agent is placed on the trans side of the bilayer, the applied potential will allow the directional control of the flow of the chaotropic agent through the pore that, ultimately, also will control the unfolding and refolding of the protein attached to the pore. This will allow investigations of reversible unfolding processes far from equilibrium at the single molecule level for the first time.