Biological nanopores are known to interact with synthetic and biological polymers, enabling their use in label-free single-molecule analytical tasks such as sequencing and/or mass discrimination. The latter, called nanopore-based single molecule mass spectrometry (Np-SMMS) has, to date, only been shown for one synthetic polymer, poly(ethyleneglycol) (PEG). It is based on the fact that binding of PEG inside the pore gives rise to blocks of ionic current, with the degree of block being detectably different for PEG molecules differing in size by only one monomer1,2. In order to extend the range of application of Np-SMMS, and to obtain information on the mechanism underying mass sensitivity of polymer-induced pore block, we have begun to test the interaction of other neutral polymers with biological nanoores. Here, we report on poly(dimethylacrylamide) (PDMA), a water-soluble neutral polymer. Under conditions used for Np-SMMS of PEG with alpha-hemolysine (4M KCl, +40 mV), we obtained current blocks with polydisperse PDMA (Mn 1500 g/mol by MALDI) that were low in frequency (<0.1 Hz/ µM) short (tau <100 µs) and noisy, resulting in little mass resolution compared to PEG. We reasoned that we might take advantage of a specific salt effect of fluoride anion reported for the aHL pore3 in order to increase blocking frequency and dwell time. Using electrolyte solutions consiting of 4M K+ as cation and various proportions of Cl- and F- as anions (1:1, 2:1, 3:1, 4:1) we were able to obtain longer events (tau up to 400 µs) and higher frequencies (up to 0.15 Hz/µM), allowing significantly better mass resolution for PDMA than in 4 M KCl. However, the large noise component in the blocked current levels for PDMA as opposed to PEG remained, still compromising the peak-to-valley ratio of histograms. These findings suggest that the specific salt effect of F- on polymer-protein interaction is independent of the polymer and may be useful in tuning polymer pore interaction for a range of analytes. (1) Robertson et al. Single-Molecule Mass Spectrometry in Solution Using a Solitary Nanopore. Proc. Natl. Acad. Sci. U S A 2007, 104, 8207-8211. (2) Baaken et al. High-Resolution Size-Discrimination of Single Nonionic Synthetic Polymers with a Highly Charged Biological Nanopore. ACS Nano 2015, 9, 6443-6449. (3) Rodrigues et al. Hofmeister Effect in Confined Spaces: Halogen Ions and Single Molecule Detection. Biophys. J. 2011, 100, 2929-2935.

Alpha-hemolysin nanopores are used to detect single oligomers of poly(ethyleneglycol) (PEG) which under high salt conditions reside in the pore for extended periods of time (up to several ms) suggesting binding to the pore's inner wall. We study the interaction of two species of PEG of degree of polymerization 28 and 32 which, following sequential entry, simultaneously reside in the pore. This doubly occupied (DO) state can result in direct replacement of the first occupant by the second. Analyzing the dwell times of the DO states, we ask whether binding as a first or second pore occupant is equivalent in terms of stability. We find that the DO state lifetimes are shorter than would be predicted by simple superposition (see Figure). We conclude that binding in the pore as the second occupant is highly unstable, suggesting that the more stable primary binding state cannot be attained by more than one PEG molecule. The doubly occupied state thus represents an intermediate state with possible general relevance for competitive interactions at binding sites to which access is gained through long channels, such as in some enzymes.

Bilayer recording is a well-established technique for in-depth studies of biophysical properties of ion channels and is particularly suited for functional studies on proteins residing in intracellular membranes. Moreover, this technique supports a host of powerful emerging analytical techniques using biological nanopores as molecular sensors. Despite its proven value, bilayer recording can be frustrating due to the capricious nature of lipid bilayers, which have to be formed manually one by one and which often lack stability. We here show a new approach and device, which speeds up the entire process by the rapid and simultaneous formation of 16, highly stable micrometer-sized bilayers using Micro-Electrode-Cavity-Array (MECA)-Chips. A study will be presented showing that the MECA supports high-resolution polymer sizing with a single biological nanopore in a parallel format (Fig.1). Additionally, data on a variety of channel proteins recorded from proteoliposomes will be shown. using a surface containing micron-sized apertures in glass substrates, the fusion of vesicles on the surface becomes an attractive method for electrophysiology and then to reconstitute membrane proteins into the lipid bilayer.

2668-Pos Board B687 Parallel and Automated Formation of Lipid Bilayers on Microstructured Chips for Ion Channel and Nanopore Recordings Gerhard Baaken1, Mohamed Kreir2, Astrid Seifert2, Matthias Beckler2, Juan M. Del Rio-Martinez1, Marcel Hoffmann1, Soenke Petersen1, Jan C. Behrends1, Niels Fertig2. University of Freiburg, Institute of Physiology, Freiburg, Germany, Nanion Technologies, München, Germany. Bilayer recording is a well-established technique for in-depth studies of biophysical properties of ion channels and is particularly suited for functional studies on proteins residing in intracellular membranes. Moreover, this technique supports a host of powerful emerging analytical techniques using biological nanopores as molecular sensors. Despite its proven value, bilayer recording can be frustrating due to the capricious nature of lipid bilayers, which have to be formed manually one by one and which often lack stability. We here show a new approach and device, which speeds up the entire process by the rapid and simultaneous formation of 16, highly stable micrometer-sized bilayers using Micro-Electrode-Cavity-Array (MECA)-Chips. A study will be presented