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Method of creating a nanopore-terminated probe for single-molecule enantiomer discrimination.

ABSTRACT: Nanopores are increasingly utilized as tools for single-molecule detection in biotechnology. Many nanopores are fabricated through procedures that require special materials, expensive facilities and experienced operators, which limit their usefulness on a wider scale. We have developed a simple method of fabricating a robust, low-noise nanopore by externally penetrating a nanocavity enclosed in the terminal of a capillary pipet. The nanocavity was shown to have a pore size on the scale of a single molecule, verified by translocation of molecules of known sizes, including double-stranded DNA (2 nm), gold nanoparticles (10 nm), and ring-shaped cyclodextrin (1.5 nm). The small pore size allows entrapment of a single cyclodextrin molecule. The glass nanopore with a trapped cyclodextrin proves useful in single-molecule discrimination of chiral enantiomers.

SUBMITTER: Gao C

PROVIDER: S-EPMC2642537 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Method of creating a nanopore-terminated probe for single-molecule enantiomer discrimination.

Gao Changlu C Ding Shu S Tan Qiulin Q Gu Li-Qun LQ

Analytical chemistry 20090101 1

Nanopores are increasingly utilized as tools for single-molecule detection in biotechnology. Many nanopores are fabricated through procedures that require special materials, expensive facilities and experienced operators, which limit their usefulness on a wider scale. We have developed a simple method of fabricating a robust, low-noise nanopore by externally penetrating a nanocavity enclosed in the terminal of a capillary pipet. The nanocavity was shown to have a pore size on the scale of a sing ...[more]

PMID: 19061410

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Project description:Prion diseases are fatal neurodegenerative diseases associated with the conversion of cellular prion protein (PrP(C)) in the central nervous system into the infectious isoform (PrP(Sc)). The mechanics of conversion are almost entirely unknown, with understanding stymied by the lack of an atomic-level structure for PrP(Sc). A number of pathogenic PrP(C) mutants exist that are characterized by an increased propensity for conversion into PrP(Sc) and that differ from wild-type by only a single amino-acid point mutation in their primary structure. These mutations are known to perturb the stability and conformational dynamics of the protein. Understanding of how this occurs may provide insight into the mechanism of PrP(C) conversion. In this work we sought to explore wild-type and pathogenic mutant prion protein structure and dynamics by analysis of the current fluctuations through an organic ?-hemolysin nanometer-scale pore (nanopore) in which a single prion protein has been captured electrophoretically. In doing this, we find that wild-type and D178N mutant PrP(C), (a PrP(C) mutant associated with both Fatal Familial Insomnia and Creutzfeldt-Jakob disease), exhibit easily distinguishable current signatures and kinetics inside the pore and we further demonstrate, with the use of Hidden Markov Model signal processing, accurate discrimination between these two proteins at the single molecule level based on the kinetics of a single PrP(C) capture event. Moreover, we present a four-state model to describe wild-type PrP(C) kinetics in the pore as a first step in our investigation on characterizing the differences in kinetics and conformational dynamics between wild-type and D178N mutant PrP(C). These results demonstrate the potential of nanopore analysis for highly sensitive, real-time protein and small molecule detection based on single molecule kinetics inside a nanopore, and show the utility of this technique as an assay to probe differences in stability between wild-type and mutant prion proteins at the single molecule level.

Dataset Information

Method of creating a nanopore-terminated probe for single-molecule enantiomer discrimination.

Publications

Method of creating a nanopore-terminated probe for single-molecule enantiomer discrimination.

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