Single molecule observation of hard–soft-acid–base (HSAB) interaction in engineeredMycobacterium smegmatisporin A (MspA) nanopores

Abstract

The principle of hard–soft-acid–base (HSAB) theory was first validated in single molecule by measurements with engineered Mycobacterium smegmatis porin A (MspA) nanopore reactors.

Rapid and multiplex preparation of engineered Mycobacterium smegmatis porin A (MspA) nanopores for single molecule sensing and sequencing

“…Though these specially designed nanoreactors are capable of resolving formation and breaking of an individual chemical bond, the reported event amplitudes are generally low, indicating a limited resolution to further resolve more refined chemical processes. MspAs were as well engineered as nanoreactors 19,20 , with which a much-enlarged event amplitude was reported, acknowledging its conical structure which serves to amplify subtle perturbations caused by single molecule reactions happening in the vicinity of the pore constriction. To further develop this technique, a significant effort in the screening of a variety of heavily mutated MspA is urgently needed 20 .…”

“…MspAs were as well engineered as nanoreactors 19,20 , with which a much-enlarged event amplitude was reported, acknowledging its conical structure which serves to amplify subtle perturbations caused by single molecule reactions happening in the vicinity of the pore constriction. To further develop this technique, a significant effort in the screening of a variety of heavily mutated MspA is urgently needed 20 . However, to date, there is no report of a method that rapidly prepares MspA mutants in a highly multiplexed way so that a variety of MspA mutants may be prepared in parallel.…”

“…However, this strategy reports a low yield and may include interferences from other non-desired channel proteins during a downstream nanopore measurement. As a recombinant protein, MspA was also expressed by E. Coli and subsequently purified by anion exchange chromatography 21 or nickel affinity chromatography 19,20 . Whereas this strategy requires the use of fast protein liquid chromatography (FPLC) instrument for purification.…”

Real-time sensing of neurotransmitters by functionalized nanopores embedded in a single live cell

“…With a short and narrow channel constriction region, MspA nanopore is a promising single-molecule analytical device. Recently, mutant MspA has been used for observing single molecule chemistry and hard-soft-acid-base interaction [38,39]. Furthermore, MspA is utilized to assist clinical diagnosis such as detection of circulating tumor cells and rapid assay for carbapenem-resistant Klebsiella pneumoniae [40,41].…”

Four Aspects about Solid‐State Nanopores for Protein Sensing: Fabrication, Sensitivity, Selectivity, and Durability

“…[ 1 ] Over the past decades, single‐molecule biosensors, which investigate one individual biomolecule per time, set themselves apart from the conventional ensemble‐averaged measurements and have gained more attention. [ 2 ] Nanopore sensors are a relatively new class of single‐molecule biosensors, which have emerged and been rapidly developed as a high‐throughput, low‐cost, analytical method for characterization of ions, [ 3–5 ] peptides, [ 6,7 ] proteins, [ 8–12 ] protein complexes, [ 13,14 ] aggregation‐prone proteins, [ 15–19 ] and nucleic acids since 1996. [ 20 ] The natural biological nanopores embedded in lipid membranes and man‐made (or solid‐state) nanopores, either fabricated in inorganic membrane or pulled using nanopipettes, constitute the core of nanopore‐based platforms.…”