No abstract

-In nature, ion conducting nanopores play a vital role for the function of living cells. They undergo gating processes where they open and close upon an external stimulus, such as the presence of a particular biomolecule, the ligand. When the gating process is observed and is quantitatively measured one can derive data about the presence and the amount of the ligand. Hence, the nanopores can be utilized for specific sensing. However, biological nanopores are embedded in a biological cell membrane that is fragile and unstable with respect to storage and application. The iNAPO project aims at combining robust polymer-based nanopores with protein-based biological nanopores, thus combining the selectivity and sensitivity of the latter with the stability and processability of the first ones. This paper describes the different steps in the fabrication of ion conducting nanopores. It begins with ion irradiation of polymer foils, combined with chemical etching of the ion damage tracks into nanopores. By means of chemical coupling reactions, the nanopore walls are functionalized with particular molecules which react or bioconjugate with the molecules to be analyzed. As an example, a recent result on sensing a physiologically active P-based anion is shown. By means of a complexation reaction with Zn-di(picolyl)amine, the selective measurement of the concentration of the anion pyrophosphate is demonstrated. In the final step of the project, the nanopores will be incorporated into a Lab-on-Chip system for applications in e.g. medical diagnostics and environmental analysis.

In nature, ion conducting nanopores play a vital role for the function of living cells. They undergo gating processes where they open and close upon an external stimulus, such as the presence of a particular biomolecule, the ligand. When the gating process is observed and is quantitatively measured, one can derive data about the presence and the amount of the ligand. Hence, the nanopores can be utilized for specific sensing. However, biological nanopores are embedded in a biological cell membrane that is fragile and unstable with respect to storage and application. The iNAPO (ion conducting nanopores) project aims at combining robust polymer-based nanopores with protein-based biological nanopores, thus combining the selectivity and sensitivity of the latter with the stability and processibility of the first ones. This paper describes the different steps in the fabrication of ion conducting nanopores. It begins with ion irradiation of polymer foils, combined with chemical etching of the ion damage tracks into nanopores. By means of chemical coupling reactions, the nanopore walls are functionalized with particular molecules which react or bioconjugate with the molecules to be analyzed. As an example, a recent result on sensing a physiologically active phosphorus-based anion is shown. By means of a complexation reaction with Zn-di(picolyl)amine, the selective measurement of the concentration of the anion pyrophosphate is demonstrated. In the final step of the project, the nanopores will be incorporated into a Lab-on-Chip system for applications in e.g. medical diagnostics and environmental analysis.

Combining the stability of solid-state nanopores with the unique sensing properties of biological components in a miniaturized electrical hybrid nanopore device is a challenging approach to advance the sensitivity and selectivity of small-molecule detection in healthcare and environment analytics. Here, we demonstrate a simple method to design an electrical hybrid nanosensor comprising a bacterial binding protein tethered to a solid-state nanopore allowing high-affinity detection of phosphonates. The diverse family of bacterial substrate-binding proteins (SBPs) binds specifically and efficiently to various substances and has been implicated as an ideal biorecognition element for analyte detection in the design of hybrid bionanosensors. Here, we demonstrate that the coupling of the purified phosphonate binding protein PhnD via primary amines to the reactive NHS groups of P(DMAA-co-NMAS) polymers inside a single track-etched nanopore in poly(ethylene terephthalate) (PET) foils results in ligand-specific and concentration-dependent changes in the nanopore current. Application of the phosphonate 2-aminoethylphosphonate (2AEP) or ethylphosphonate (EP) induces a large conformational rearrangement in PnhD around the hinge in a venus flytrap mechanism resulting in a concentration depended on increase of the single pore current with binding affinities of 27 and 373 nM, respectively. Thus, the specificity and stability of this simple hybrid sensor concept combine the advantages of both, the diversity of ligand-specific substrate-binding proteins and solid-state nanopores encouraging further options to produce robust devices amenable to medical or environmental high-throughput-based applications in nanotechnology.

The glycine receptor (GlyR), a member of the pentameric ligand-gated ion channel family (pLGIC), displays remarkable variations in the affinity and efficacy of the full agonist glycine and the partial agonist taurine depending on the cell system used. Despite detailed insights in the GlyR three-dimensional structure and activation mechanism, little is known about conformational rearrangements induced by these agonists. Here, we characterized the conformational states of the α1 GlyR upon binding of glycine and taurine by microscale thermophoresis expressed in HEK293 cells and Xenopus oocytes after solubilization in amphipathic styrene-maleic acid copolymer nanodiscs. Our results show that glycine and taurine induce different conformational transitions of the GlyR upon ligand binding. In contrast, the variability of agonist affinity is not mediated by an altered conformational change. Thus, our data shed light on specific agonist induced conformational features and mechanisms of pLGIC upon ligand binding determining receptor activation in native environments.