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The development of a diamond-based nanopore sensor for the detection and identification of DNA

Final Report Summary - BIONANODIAMOND (The development of a diamond-based nanopore sensor for the detection and identification of DNA)

The detection and identification of specific sequences of DNA is a critical tool in many fields\including early medical diagnostics, forensics and the rapid detection of bio-warfare agents. Despite\the enormous potential, widespread use of DNA testing is not routine in any of these applications because of the high expense and the requirement for testing to be run by highly trained personnel working in a laboratory.

Nanopores have received recent attention as one potential route to solving the aforementioned problems. In nanopore measurements, DNA molecules are captured one-at-a-time by a protein nanopore that has internal dimensions similar to that of DNA. Ion flux through the pore is
continuously measured, and from these measurements DNA can be identified entering and residing inside the pore. The precise change in ion flux is dependent on the structure of the DNA, and so, can be used to identify variation of the DNA inside the pore. The BIONANODIAMOND project aimed to exploit nanopore analysis of DNA to develop an assay capable of continuously and selectively detecting DNA of interest with very high sensitivity.

This report describes the progress made in developing nanopore technology over the three year period of the project. The first two years were based at the University of Utah, USA (outgoing phase) and focused on the development of new nanopore techniques. The final year was based at the University of Warwick, UK (return phase) and in which knowledge of the newly developed nanopore methods were transferred back to Europe, and research focussed on integrating the nanopore method into a robust device by researching the potential of diamond as a material for nanopore measurements.

Research during the outgoing phase, resulted in significant new insights into DNA capture by nanopores, and by extension, the development of new methodologies to detect important structural changes in DNA that can lead to cancer and other diseases. Of particular note is the development of novel methodologies to detect mismatches and epigenetic markers in DNA at the single molecule level. This work could, with further development, form the basis of devices capable of detecting harmful variations in DNA and have a significant social impact on the well-being of citizens by proving a method to detect cancers and other diseases more rapidly, which in turn leads to better health outcomes through earlier commencing of treatment. The commercial potential of the technology developed during the project is evidenced by the recent filing of 2 provisional patents, and the publication of 2 manuscripts in top-level chemistry Journal J. Am. Chem. Soc. These results were also disseminated via oral presentations at conferences.

The most significant discovery from a fundamental science perspective during the outgoing phase was that the biological processes of “base-flipping”, which is believed to play a role in the interaction of mutation and damaged bases with repair enzymes, could be monitored within the confined environment of the protein pore. It was possible for the first time monitor the kinetics of a base-flipping for a mismatched base-pair at the single molecule level. The techniques developed during the project have provided insights into biophysical processes that are important to DNA damage and repair. When DNA repair does not function correctly, it can lead to diseases, and thus proper understanding of these processes is critical in advancing our understanding of how to improve human health. The implications of this research have been published primarily in an article for the journal Faraday discussions.

During the incoming phase, research focused on the development of a solid-state nanopore in diamond, and the surface modification of diamond substrates. Some progress was made within both areas. With collaborators, a nanopore in diamond was fabricated and used for some initial measurements of particle translocation. It was possible to detect individual polystyrene particles translocating through the diamond nanopore, and it was discovered that the extent to which the particle transport changed the measured current was found to be dependent on the direction of travel. This work has recently been submitted to the Journal Carbon. By preparing a nanopore in diamond, it may be possible to develop nanopore sensors that significantly more robust than existing glass and silicon based pores, for which there is there potential economic benefit via the development of the more stable and reliable biosensors.

In terms of knowledge transfer into Europe, the host group at the University of Warwick has gained new capabilities in bio-sensing, surface modification and nanopore techniques, knowledge of which the research fellow bought back with him and has disseminated to PhD students and post-doctoral researchers within the UK-based group. In particular, the knowledge of how to modify surfaces with DNA is now being used in a new project to develop diamond based DNA sensors.

Finally, the BIONANODIAMOND project also solidified existing research collaborations between the Universities of Utah and Warwick, and further collaborative work between the two research groups into the future and beyond this project in the area of nanopore sensing is envisaged.