Optical Single Channel Recording for Nanopore Sensing

Genome sequencing methods have led to a rapid expansion in our understanding of the subtleties and variations in how genetic profiles affect disease. This has been spurred in part by new technological advances. Nanopore sequencing has been highlighted as one upcoming technology with the potential to go further, and improve the speed and cost effectiveness of seqencing. In nanopore sensing, the flow of ions through a nanoscopic protein or solid-state pore is disrupted by the presence of an analyte. If this analyte is DNA, blockades in the current corresponding to the sequence are detected. Long reads of many thousands of bases can be made without amplification or labelling.
Our research track record marks a long-standing interest in developing methods capable of detecting individual molecules using fluorescence microscopy. We have used this to improve our understanding of membrane protein function, and in particular, how specialized protein pores work. Most recently we used these methods to demonstrate DNA base detection in a nanopore using an optical, rather than an electrical readout. These methods have the potential to improve the parallelization of nanopore sensing.
In collaboration with Oxford Nanopore Technologies we have identified two essential challenges for current nanopore sensing where an optical readout provides significant impact: 1. We will combine optical single channel recording and single-molecule fluorescence imaging to understand and then optimise the mechanistic steps of nanopore sensing; 2. We will go beyond the current speed and sensitivity limitations of optical single channel recording and develop new single-molecule microscopy methods for nanopore sensing.

Recent advances in 'fourth-generation' genome sequencing methods have led to a dramatic reduction in the cost per base - driving a marked expansion in our understanding of the subtleties and variations in how genetic profiles affect disease. Nanopore sequencing has been highlighted as an upcoming technology with the potential to further disrupt this field. In nanopore sensing, the flow of ions through a nanoscopic protein or solid-state pore is disrupted by the presence of an analyte. If this analyte is DNA, blockades in the current corresponding to the sequence are detected. Long reads of many kilobases can be made without amplification or labelling.

Recently we used these methods to demonstrate DNA base detection in a nanopore using optical, rather than an electrical readout. These methods have the potential to improve the parallelization of nanopore sensing. In collaboration with Oxford Nanopore Technologies we have identified two essential challenges for current nanopore sensing where an optical readout provides significant impact: 1. We will combine optical single channel recording and single-molecule fluorescence imaging to understand and then optimise the mechanistic steps of nanopore sensing, from membrane binding, to capture and readout; 2. We will go beyond the current speed and sensitivity limitations of optical single channel recording by developing interferometric scattering microscopy for nanopore sensing.

These objectives address two distinct areas - improving our fundamental understanding of nanopore sensing, and pushing beyond the capabilities of current methods. They have significant synergy: By improving our understanding of the process of sensing we will accelerate our ability to develop these new methods, and our new tools will help expand our understanding of nanopore sensing.

The societal importance of applying next-generation sequencing methods to human disease is evident. Recent commercialisation of this technology has led to an 'arms race' in next gen sequencing, and subsequently holds the promise for a significant boon to our understanding of human health. The rapid commercialisation of nanopore sensing has thus far focused on DNA sequencing, however these methods hold the potential to address a range of other biological problems, including post-translational modification, protein and small molecule detection.

In the short term, academic impact would be met through interaction with our current international network of biological collaborators; and in the long term by publication and through dissemination of our work, using the path of patent protection, licensing and spin out we have followed. In particular, the new optical methods proposed here would have immediate cross-over to improve our understanding of ion-channel biology and the high-throughput screening of ion channels.

This proposal helps both to develop new methods that further the scope of nanopore sensing, and help provide a robust understanding of the mechanism of analyte capture and detection by a nanopore. This work is a strong match to the BBSRC strategic priority of 'technology development for the biosciences' leading to advances that have particular impact for 'bioscience for health'.