Gerhard Baaken

Nanopore-Based Single-Molecule Mass Spectrometry on a Lipid Membrane Microarray

We report on parallel high-resolution electrical single-molecule analysis on a chip-based nanopore microarray. Lipid bilayers of <20 μm diameter containing single alpha-hemolysin pores were formed on arrays of subpicoliter cavities containing individual microelectrodes (microelectrode cavity array, MECA), and ion conductance-based single molecule mass spectrometry was performed on mixtures of poly(ethylene glycol) molecules of different length. We thereby demonstrate the function of the MECA device as a chip-based platform for array-format nanopore recordings with a resolution at least equal to that of established single microbilayer supports. We conclude that devices based on MECAs may enable more widespread analytical use of nanopores by providing the high throughput and ease of operation of a high-density array format while maintaining or exceeding the precision of state-of-the-art microbilayer recordings.

High-Resolution Size-Discrimination of Single Nonionic Synthetic Polymers with a Highly Charged Biological Nanopore

Electrophysiological studies of the interaction of polymers with pores formed by bacterial toxins (1) provide a window on single molecule interaction with proteins in real time, (2) report on the behavior of macromolecules in confinement, and (3) enable label-free single molecule sensing. Using pores formed by the staphylococcal toxin α-hemolysin (aHL), a particularly pertinent observation was that, under high salt conditions (3-4 M KCl), the current through the pore is blocked for periods of hundreds of microseconds to milliseconds by poly(ethylene glycol) (PEG) oligomers (degree of polymerization approximately 10-60). Notably, this block showed monomeric sensitivity on the degree of polymerization of individual oligomers, allowing the construction of size or mass spectra from the residual current values. Here, we show that the current through the pore formed by aerolysin (AeL) from Aeromonas hydrophila is also blocked by PEG but with drastic differences in the voltage-dependence of the interaction. In contrast to aHL, AeL strongly binds PEG at high transmembrane voltages. This fact, which is likely related to AeLs highly charged pore wall, allows discrimination of polymer sizes with particularly high resolution. Multiple applications are now conceivable with this pore to screen various nonionic or charged polymers.

Automated Formation of Lipid Membrane Microarrays for Ionic Single-Molecule Sensing with Protein Nanopores

Efficient use of membrane protein nanopores in ionic single-molecule sensing requires technology for the reliable formation of suspended molecular membranes densely arrayed in formats that allow high-resolution electrical recording. Here, automated formation of bimolecular lipid layers is shown using a simple process where a poly(tetrafluoroethylene)-coated magnetic bar is remotely actuated to perform a turning motion, thereby spreading phospholipid in organic solvent on a nonpolar surface containing a <1 mm(2) 4 × 4 array of apertures with embedded microelectrodes (microelectrode cavity array). Parallel and high-resolution single-molecule detection by single nanopores is demonstrated on the resulting bilayer arrays, which are shown to form by a classical but very rapid self-assembly process. The technique provides a robust and scalable solution for the problem of reliable, automated formation of multiple independent lipid bilayers in a dense microarray format, while preserving the favorable electrical properties of the microelectrode cavity array.

Characteristics of highly flexible PDMS membranes for long-term mechanostimulation of biological tissue.

Measurement of mechanical properties of soft biological tissue remains a challenging task in mechanobiology. Recently, we presented a bioreactor for simultaneous mechanostimulation and analysis of the mechanical properties of soft biological tissue samples. In this bioreactor, the sample is stretched via deflection of a flexible membrane. It was found that the use of highly compliant membranes increases accuracy of measurements. Here, we describe the production process and characteristics of thin and flexible membranes of polydimethylsiloxane (PDMS) designed to improve the signal-to-noise ratio of our bioreactor. By a spin-coating process, PDMS membranes were built by polymerization of a two component elastomer. The influence of resin components proportion, rotation duration, and speed of the spinning were related to the membrane mechanics. Membranes of 22 mm inner diameter and 33 to 36 microm thickness at homogeneous profiles were produced. Isolated rat diaphragms served as biological tissue samples. Mechanical properties of the membranes remained constant during 24 h of mechanostimulation. In contrast, time- and strain-dependent mechanical properties of the diaphragms were found.

Translocation of Precision Polymers through Biological Nanopores

Nanopore analysis, which is, currently, chiefly used for DNA sequencing, is also an appealing technique for characterizing abiotic polymers. As a first step toward this goal, nanopore detection of non-natural monodispersed poly(phosphodiester)s as candidate backbone structures is reported herein. Two model homopolymers containing phosphopropyl repeat units (i.e., 56 or 104 r.u.) and a short thymidine nucleotide sequence are analyzed in the present work. They are tested in two different biological nanopores, α-hemolysin from Staphylococcus aureus, and aerolysin from Aeromonas hydrophila. These recordings are performed in aqueous medium at different KCl concentrations and various driving voltages. The data show a complex interaction with evidence for voltage dependence and threading, and underline the influence of the molecular structure and orientation of the precision poly(phosphodiester)s on the observed residual current signal as well as on the translocation dynamics. In particular, they suggest a dominant entropic contribution due to the high flexibility of the phosphodiester homopolymer.

Hochauflösende Einzelmolekülanalyse mit Nanoporen-Arrays

ZusammenfassungEin Array aus einzelnen individuell elektrisch kontaktierten biologischen Nanoporen in synthetischen Lipidmembranen erlaubt die parallele Detektion einzelner Moleküle in wässrigen Lösungen. Die hohe Auflösung der Messungen wird beim Einsatz zur präzisen Bestimmung der Massenverteilung von Polymeren deutlich.AbstractA chip array in which single biological nanopores in synthetic lipid membranes are electrically contacted individually allows parallel single molecule detection in aqueous solution. The high resolution of the measurements is shown in an application to precisely determine the distribution of polymer masses.

Size-dependent interaction of a 3-arm star poly(ethylene glycol) with two biological nanopores

Abstract.We use two pore-forming proteins, alpha-hemolysin and aerolysin, to compare the polymer size-dependence of ionic current block by two types of ethyleneglycol polymers: 1) linear and 2) 3-arm star poly(ethylene glycol), both applied as a polydisperse mixture of average mass 1kDa under high salt conditions. The results demonstrate that monomer size sensitivity, as known for linear PEGs, is conserved for the star polymers with only subtle differences in the dependence of the residual conductance on monomer number. To explain this absence of a dominant effect of polymer architecture, we propose that PEG adsorbs to the inner pore wall in a collapsed, salted-out state, likely due to the effect of hydrophobic residues in the pore wall on the availability of water for hydration.Graphical abstract

Parallel, High-resolution Nanopore Analysis on a Chip-based Lipid Membrane Micorarray

In order to be used as single-molecule-sensitive analyte detectors1-3, biological nanopores need to be functionally reconstituted into free-standing synthetic membranes between two electrolyte compartments that can be contacted by electrodes. Classically, phospholipid bilayers are spread over an opening in a septum between two compartments filled with salt solution and connected by means of non-polarizable electrodes (Ag/AgCl) with the measurement electronics. The set-ups and methods used in current research for such measurements can only be used productively by experienced specialists. They are not amenable to automation and do not support simultaneous measurements at multiple single pores. Future exploitation of the manifold and attractive applications for nanopore analytics in Chemistry and Biology will depend crucially on the development of platform technologies that allow rapid and, if possible, automatic electrical measurements for nanopores with maximally enhanced throughput.

High Resolution Single Molecule Analysis using Nanopore Recording on Microelectrode Cavity Arrays

Single molecule detection using biological nanopores in lipid bilayers is crucially limited by noise and bandwidth of the recording. We have tested a newly developed 16-channel microelectrode cavity array (MECA, Ref. 1) for single molecule detection using alpha-Hemolysin (alphaHL) nanopores. The device is based on subpicoliter cavities in a high-quality dielectric polymer adding less than 0.5 pF to the input capacitance of the amplifier (Axopach200B), thereby optimizing noise and bandwidth.View Large Image | View Hi-Res Image | Download PowerPoint SlideAn example trace is shown in Fig.1A with an open state rms noise of 0.65pA at 0-5 kHz. Note that for the blocked state, there is no significant difference between an all points histogram of a 5 kHz filtered trace (black) and that of a event amplitudes defined by averaging (grey). Fig.1B shows 35 single PEG induced blockages superimposed and aligned in time. They are detected as square pulses down to durations<100µs and longer events seem to correlate with deeper blocks. The recording performance of MECAs with high integration densities and superior mechanical stability is expected to greatly facilitate single molecule nanopore analysis in the future.(1)Baaken et al.(2008),Lab Chip 8(6):938-44.