Oxford Nanopore's first generation of technology uses bespoke, proprietary pore-forming proteins to create pores in membranes. Pore-forming proteins are common in nature.
For example, the protein α-hemolysin and 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.
α-hemolysin is a heptameric protein pore with an inner diameter of 1 nm, 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 and our collaborators. The Company has optimised the large-scale production of this and many other bespoke pore-forming proteins, each of which have different characteristics suitable for different applications. Oxford Nanopore is continuously investigating new nanopores with new properties that can improve product performance.
Adaptation of protein nanopores for the identification of single molecules
Protein nanopores can be adapted at Angstrom-level precision using protein-engineering techniques. Specific adaptations can be designed so that the nanopore is a sensor for a range of specific molecules. Techniques include:
- Changing the architecture of the internal structure of the nanopore so that it affects the passage of an analyte through the pore.
- The incorporation of a DNA probe to detect an organism with the matching DNA code.
- The attachment of a molecular motor – for example a processive enzyme – for the analysis of polymers such as DNA.
- The attachments of ligands/aptamers to the nanopore, to bind with target proteins outside the pore.
Oxford Nanopore has developed proprietary methods for the rapid design and production of bespoke nanopores, by programmed bacteria. The Company continuously develops and releases new nanopores and adaptations of those nanopores, towards improving product performance.
The Company has also developed proprietary electronics that enable multiple nanopore sensing experiments to be performed in parallel, the data collected, and analysed in real time. These devices can be scaled according to need; read about the handheld MinION or the desktop PromethION.
Future generations of nanopores: solid state
Protein nanopores are robust, easily reproducible at low cost, and easy to modify. However, future generations of nanopore sensing devices are likely to use nanopores fabricated from synthetic materials: solid-state nanopores. These have the potential to improve the cost and scale of nanopore analyses even further.
Oxford Nanopore has internal R&D projects and collaborations with research groups, developing innovative solid-state nanopore technologies into a next-generation nanopore technology. These collaborations include a broad intellectual property estate for solid-state nanopore sensing platforms in a variety of forms including silicon nitride, graphene, and modifications to these solid-state materials for the sensing process.
In 2008, Oxford Nanopore established a collaboration with the laboratories of Professors Daniel Branton and Jene Golovchenko at Harvard University, early pioneers of nanopore sensing, and particularly in the development of methods of solid-state sequencing. Oxford Nanopore supports research in these laboratories and licenses the right to develop nanopore discoveries into a single molecule analysis technology. In 2011, a further collaboration was announced between Harvard and Oxford Nanopore, for the development of graphene as a solid-state nanopore sequencing device.
A solid-state nanopore is typically a nanometer-sized hole formed in a synthetic membrane (usually SiNx or SiO2). The pore is usually fabricated by focused ion or electron beams, so the size of the pore can be tuned freely, although further development is necessary to reach the atomic precision naturally achieved by protein pores. Because of the ability to tune pore geometry, and the superior mechanical and chemical stability of solid-state membranes, considerable R&D work has been performed in this field, including alternative sequencing/diagnostic strategies, new membrane materials, hybrid pores and integrated sensors.
Solid-state nanopores currently lack the chemical specificity of protein nanopores. A method under exploration is the integration of a protein pore into a solid-state membrane.
Nanopores with integrated sensors
Integrated sensors have been explored as technologies to supersede methods involving ionic current measurement. Proposed techniques include tunnelling electrode-based detectors, capacitive detectors and graphene-based nano-gap or edge state detectors. Recently, the local voltage signal generated by DNA translocation – which is proportional to ionic current signal – has been detected experimentally by transistors. This detection scheme may be an attractive alternative to ionic current because it preserves the information of ionic current signal with the potential to achieve much higher integration density and higher speed.
Graphene is a robust, single-atom-thick ‘honeycomb’ lattice of carbon with high electrical conductivity. These properties make it an ideal material for high resolution, nanopore-based sequencing of single DNA molecules. The fine depth of the graphene membrane provides optimal spatial resolution along the DNA, and at the same time, graphene is extremely strong and chemically inert. Graphene itself is also a good electronic sensor material, which is sensitive to nearby molecules and the chemical/electrical environment.
In a landmark publication Garaj S et al., Nature 467 (7312), 190–193 (2010) the Branton and Golovchenko teams used graphene to separate two chambers containing ionic solutions, and created a nanopore in the graphene. The group demonstrated that the graphene nanopore could be used as a trans-electrode, measuring a current flowing through the nanopore between two chambers. The trans electrode was used to measure variations in the current as a single molecule of DNA was passed through the nanopore. This resulted in a characteristic electrical signal that reflected the size and conformation of the DNA molecule.
At one atom thick, graphene is believed to be the thinnest membrane able to separate two liquid compartments from each other. This is an important characteristic for DNA sequencing; a trans electrode of this thickness would be suitable for the accurate analysis of individual bases on a DNA polymer as it passes through the graphene. Further developments are required to make high-quality graphene pores with precise structure and edge chemistry that would be required for direct label-free sequencing.
Oxford Nanopore's in-house solid-state sequencing project is based in Cambridge, MA, USA.