Enzyme Sandwich of Planar Unsupported Porous Polymer-Lipid Hybrid Bilayer for Single-Molecule Nanopore Enzymology

Electrochemical recording of traditional planar unsupported lipid bilayer is crucial for screening ion channel blocking drugs and studying the biophysical properties of protein channels. However, the fragility and instability of lipid bilayer limit their development and applications. In this work, we developed a robust polydopamine (PDA)-lipid hybrid bilayer platform, which allows membrane proteins to easily insert and form stable channels. Furthermore, individual enzyme molecules can be tightly confined between a porous polymer aerogel film and a lipid monolayer to enable a nanopore-based enzyme sandwich assay. We demonstrated that alkaline phosphatases (ALP) could be analyzed through confinement effects. The nanopore current fluctuations revealed six conformational states of ALP, including three ground-state and three enzyme-substrate/inhibitor bound conformations. The catalytic kinetics of ALP were directly characterized by monitoring the enzyme-substrate interaction without any chemical bonding, modification, or labeling.