Amplification of Single Molecule Translocation Signal Using β‑Strand Peptide Functionalized Nanopores

Changes in ionic current flowing through nanopores due to binding or translocation of single biopolymer molecules enable their detection and characterization. It is, however, much more challenging to detect small molecules due to their rapid and small signal signature. Here we demonstrate the use of <i>de novo</i> designed peptides for functionalization of nanopores that enable the detection of a small analytes at the single molecule level. The detection relies on cooperative peptide conformational change that is induced by the binding of the small molecule to a receptor domain on the peptide. This change results in alteration of the nanopore effective diameter and hence induces current perturbation signal. On the basis of this approach, we demonstrate here the detection of diethyl 4-nitrophenyl phosphate (paraoxon), a poisonous organophosphate molecule. Paraoxon binding is induced by the incorporation of the catalytic triad of acetylcholine esterase in the hydrophilic domain of a short amphiphilic peptide and promotes β-sheet assembly of the peptide both in solution and for peptide molecules immobilized on solid surfaces. Nanopores coated with this peptide allowed the detection of paraoxon at the single molecule level revealing two binding arrangements. This unique approach, hence, provides the ability to study interactions of small molecules with the corresponding engineered receptors at the single molecule level. Furthermore, the suggested versatile platform may be used for the development of highly sensitive small analytes sensors

Fabrication of nanopores in multi-layered silicon-based membranes using focused electron beam induced etching with XeF₂ gas.

The emergent technology of using nanopores for stochastic sensing of biomolecules introduces a demand for the development of simple fabrication methodologies of nanopores in solid state membranes. This process becomes particularly challenging when membranes of composite layer architecture are involved. To overcome this challenge we have employed a focused electron beam induced chemical etching process. We present here the fabrication of nanopores in silicon-on-insulator based membranes in a single step process. In this process, chemical etching of the membrane materials by XeF2 gas is locally accelerated by an electron beam, resulting in local etching, with a top membrane oxide layer preventing delocalized etching of the silicon underneath. Nanopores with a funnel or conical, 3-dimensional (3D) shape can be fabricated, depending on the duration of exposure to XeF2, and their diameter is dominated by the time of exposure to the electron beam. The demonstrated ability to form high-aspect ratio nanopores in comparably thick, multi-layered silicon based membranes allows for an easy integration into current silicon process technology and hence is attractive for implementation in biosensing lab-on-chip fabrication technologies