Nanopores: biosensors of single molecules

Research into nanopores has been conducted at leading academic institutions worldwide for nearly 20 years, revealing their unique capacity to detect and analyse specific molecules, i.e. to act as biosensors. 

Oxford Nanopore was founded to develop this technology into a platform with broad commercial applications. Existing methods of detecting single molecules are complex and expensive, particularly where they involve optical detection. A method that is direct and electrical has the potential to deliver greater power, improved cost, greater versatility and improved workflow over existing solutions.

About nanopores

A nanopore is, essentially, a very small hole. This hole may be formed either by a protein pore set into a membrane (biological nanopores), or by artificially creating a hole in solid materials (solid state nanopores).

A nanopore may be used to identify a target analyte in the following way: 

A current is passed through the nanopore.  If an analyte passes through the pore or past its aperture, this event creates a characteristic disruption in current.  By measuring that current it is possible to identify the molecule in question. For example, this system can be used to distinguish the four standard DNA bases and G, A, T and C, and also methylated C.  It can be used to tell the difference between enantiomers of ibuprofen or to identify reactive compounds such as explosives.

Alpha-hemolysin

Alpha-hemolysin mutant to facilitate a molecular adapter for improved analyte detection and discrimination. This nanopore is shown with a custom cyclodextrin providing the binding site.

Oxford Nanopore's first generation of technology uses the protein nanopore alpha-hemolysin.  Similar protein pores are found naturally in cell membranes, where they act as channels for ions or molecules to be transported in and out of cells. For example, the bacteria S. aureus produces protein nanopores such as alpha-hemolysin as a tool to extract the contents of the cells of other organisms. 

Alpha-hemolysin is a heptameric protein pore with an inner diameter of 1nm, about 100,000 times smaller than that of a human hair. This diameter is the same scale as many single molecules, including DNA. The pore is highly stable and has been characterised in great detail by Oxford Nanopore's collaborators and researchers elsewhere. The Company has optimised the production of this protein in a wide range of expression systems, enabling the large-scale, consistent manufacture of wild type and genetically modified forms of alpha-hemolysin. 

Adaption of alpha-hemolysin for the identification of single molecules

The α-hemolysin nanopore can be adapted using protein engineering techniques, to be a sensor for a range of specific molecules. This can be done in a variety of ways including:

• The incorporation of a specific binding site within the nanopore, that will bind transiently with the molecule being detected. This might be a cyclodextrin in the case of DNA base, or other adapters for other small molecules.
• The incorporation of a DNA probe to detect an organism with the matching DNA code
• The attachments of ligands to the nanopore, to bind with target proteins outside the pore

For a full explanation of Oxford Nanopore's first generation of DNA sequencing, click here
For a list of relevant publications please click here for the publications page.
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