We introduce a two-dimensional method for mass spectrometry in solution that is based on the interaction between a nanometer-scale pore and analytes. As an example, poly(ethylene glycol) molecules that enter a single ␣-hemolysin pore cause distinct mass-dependent conductance states with characteristic mean residence times. The conductance-based mass spectrum clearly resolves the repeat unit of ethylene glycol, and the mean residence time increases monotonically with the poly(ethylene glycol) mass. This technique could prove useful for the real-time characterization of molecules in solution.Gaussian mixture model ͉ poly(ethylene glycol) ͉ ␣-hemolysin ͉ ion channel ͉ maximum likelihood signal classification

Nanometer-scale pores have demonstrated potential for the electrical detection, quantification, and characterization of molecules for biomedical applications and the chemical analysis of polymers. Despite extensive research in the nanopore sensing field, there is a paucity of theoretical models that incorporate the interactions between chemicals (i.e., solute, solvent, analyte, and nanopore). Here, we develop a model that simultaneously describes both the current blockade depth and residence times caused by individual poly(ethylene glycol) (PEG) molecules in a single α-hemolysin ion channel. Modeling polymer-cation binding leads to a description of two significant effects: a reduction in the mobile cation concentration inside the pore and an increase in the affinity between the polymer and the pore. The model was used to estimate the free energy of formation for K þ -PEG inside the nanopore (≈ − 49.7 meV) and the free energy of PEG partitioning into the nanopore (≈0.76 meV per ethylene glycol monomer). The results suggest that rational, physical models for the analysis of analyte-nanopore interactions will develop the full potential of nanopore-based sensing for chemical and biological applications.alpha-hemolysin | nanopore-based sensing | polymer confinement | polymer analysis P olymers play a fundamental role in life (1) and are central to many emerging technologies (2). Many of these applications require a detailed understanding of the structure, morphology, and chemical interactions of polymers under confinement in either 2-dimensional films (3) or narrow tubes (4). The ability to isolate and study single molecules has shown promise in overcoming the limitations of measurements with ensemble averages and permits probing the inter-and intra-molecular forces, structural changes, and dynamics of polymers (for a detailed review of single-molecule polymer analysis see refs. 5 and 6).Molecules partition into a nanopore and alter the flow of ions resulting in distinct current blockades that can be used to detect, characterize, and quantify a wide range of polymer types (7). These include single-stranded RNA and DNA (8-10), proteins (11-14), biowarfare agents (15), therapeutic agents against anthrax toxins (15, 16), and chemically synthesized molecules (14,(17)(18)(19)(20). More recently, single nanopores were used to determine the size distribution of polymers in a manner akin to mass spectrometry (19).To fully realize the potential of nanopore-based sensors, it is important to develop a detailed understanding of the physical and chemical interactions of polymers with the nanopore, solvent, electrolyte, and other components. In this work, the interaction between poly(ethylene glycol) (PEG) and the α-hemolysin (αHL) channel in a high ionic strength electrolyte was used to develop a unique model of polymers confined within single nanopores.Previous attempts to describe the magnitude of PEG-induced nanopore current blockades focused on volume exclusion (21) and/or microviscosity (22), which required adjustable ad hoc...

There are thousands of different nanometer-scale pores in biology, many of which act as sensors for specific chemical agents. Recent work suggests that protein and solid-state nanopores have many potential uses in a wide variety of analytical applications. In this review we survey this field of research and discuss the prospects for advances that could be made in the near future.

We demonstrate a method for simultaneous structure and function determination of integral membrane proteins. Electrical impedance spectroscopy shows that Staphylococcus aureus alpha-hemolysin channels in membranes tethered to gold have the same properties as those formed in free-standing bilayer lipid membranes. Neutron reflectometry provides high-resolution structural information on the interaction between the channel and the disordered membrane, validating predictions based on the channel's x-ray crystal structure. The robust nature of the membrane enabled the precise localization of the protein within 1.1 A. The channel's extramembranous cap domain affects the lipid headgroup region and the alkyl chains in the outer membrane leaflet and significantly dehydrates the headgroups. The results suggest that this technique could be used to elucidate molecular details of the association of other proteins with membranes and may provide structural information on domain organization and stimuli-responsive reorganization for transmembrane proteins in membrane mimics.

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