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Highly Engineered Nanopores for Protein Detection: Single-Molecule Sensing Under Extreme Conditions

This research project focused on mechanically stabilizing the FhuA-based nanopore for protein detection, resulting in a robust and noise-free nanopore. The engineered nanopore, termed ΔC/Δ4L, was open and stable, allowing for biomolecular interaction sensing and pH-induced digestion of proteins. The study showcased the nanopore's viability as a biosensor for single-molecule stochastic sensing under varying experimental conditions, expanding its potential applications in fields like drug delivery and synthetic biology.

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Highly Engineered Nanopores for Protein Detection: Single-Molecule Sensing Under Extreme Conditions

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  1. Single-Molecule Detection of ProteinsLiviu Movileanu, Syracuse University, DMR 1006332 The major goal of this research project was the mechanical stabilization of the highly engineered FhuA-based nanopore C/4L. By coupling techniques in membrane protein engineering and specific protein refolding protocols, we were able to produce a nanopore that was exceptionally quiet and robust under a broad range of experimental circumstances. The systematic removal of pore-occluding structures in the native FhuA resulted in C/4L, a clean open nanopore. The application of a detergent-based fast protein refolding protocol resulted in a rigid protein scaffold under harsh conditions of experimentation. This work is now published in Journal of the American Chemical Sociery134 (2012) 9521-9531. In this work, the mechanically stable FhuA C/4L nanopore has demonstrated the ability to sense biomolecular interactions and the pH induced digestion of IgG by pepsin under conditions not yet achievable with other protein nanopores. A 3.1 nm 4.4 nm B ±120 mV 20 mM KCl 2.5 s Current (pA) pH 11.6 pH 2.8 Figure A shows the native FhuA protein in the left panel with a model of the highly engineered C/4L on the right. FigureB illustrates the unique robustness of the open-state current of the C/4L nanopore over a range of voltages and pH values. pH 4.3 Applied potential (mV)

  2. Single-Molecule Detection of ProteinsLiviu Movileanu, Syracuse University, DMR 1006332 Education: Researchers that contributed significantly to these results include several outstanding undergraduate students, such as Manu Arul, a Beckman Scholar pictured here, Katie Rosiene, Josh Mills, and Jeffrey Roberge. Jeffrey is a recipient of aNSF-funded REU Biomaterials internship. The current undergraduate researchers were fully engaged in every aspect of our group. Their laboratory experience was integrated with the graduate research mentors, including Aaron J Wolfe, David J Niedzwiecki and Belete R Cheneke. Impact: Here, we have shown that these protein nanopores could be used as integral elements of biosensors for single-molecule stochastic sensing under conditions of low physiological salt concentration and extreme pH values. These results expand the potential for utilizing the C/4L engineered protein nanopores in various arenas, including separation-based science, drug delivery, synthetic biology and biotherapeutics.

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