Evaluation of Nanopore Sensor Design Using Electrical and Optical Analyses

Abstract

Nanopores are currently utilized as powerful tools for single-molecule protein sensing. The reporting signal typically requires protein analytes to enter the nanopore interior, yet a class of these sensors has emerged that allows targeted detection free in solution. This tactic eliminates the spatial limitation of nanopore confinement. However, probing proteins outside the nanopore implies numerous challenges associated with transducing the physical interactions in the aqueous phase into a reliable electrical signature. Hence, it necessitates extensive engineering and tedious optimization routes. These obstacles have prevented the widespread adoption of these sensors. Here, we provide an experimental strategy by developing and validating single-polypeptide-chain nanopores amenable to single-molecule and bulk-phase protein detection approaches. We utilize protein engineering, as well as nanopore and nanodisc technologies, to create nanopore sensors that can be integrated with an optical platform in addition to traditional electrical recordings. Using the optical modality over an ensemble of detectors accelerates these sensors’ optimization process for a specific task. It also provides insights into how the construction of these single-molecule nanopore sensors influences their performance. These outcomes form a basis for evaluating engineered nanopores beyond the fundamental limits of the resistive-pulse technique.

Real-Time Measurement of a Weak Interaction of a Transcription Factor Motif with a Protein Hub at Single-Molecule Precision

“…The omll4tfhua gene was purchased from GenScript (Piscataway, NJ) . At the N terminus, each gene also included a sequence encoding a peptide ligand for interacting with WDR5 through one of its binding sites and a 13-residue peptide adaptor (O, MGDRGPEFELGTM) . The mll4 gene encoded a 14-residue M ixed L ineage L eukemia 4 (MLL4) W DR6 in teraction (Win) motif peptide ligand (MLL4 Win , LNPHGAARAEVYLR). , A site-directed mutagenesis kit (New England Biolab, Ipswich, MA) was utilized to substitute the mll4 gene with the myc gene.…”

“…Before reconstitution, the nanodiscs (NDs) were fabricated from biotinylated membrane scaffold proteins (MSP). , The reconstitution of MYC WBM tFhuA occurred in one step. The detergent-solubilized MYCWBMtFhuA was combined with biotinylated MSP at a ratio of 1:2, respectively.…”

Gating of β-Barrel Protein Pores, Porins, and Channels: An Old Problem with New Facets

“…This relatively large single-channel conductance results from the passage of ions across an elliptical internal pore with sides of 2.6 × 3.6 nm. This FhuA variant was frequently employed for further developments in biosensing applications because of its monomeric nature, high thermodynamic stability, and relatively larger size [150][151][152][153][154][155][156].…”