In situ extraction and detection of DNA is an im-pore-tant development

Osaka – Being able to detect DNA from a single cell is important for the detection of diseases and genetic disorders. Measuring single DNA molecules has been possible for some time; however, directly detecting samples at the point of extraction with no need for subsequent steps has not. Now, researchers at SANKEN, Osaka University have demonstrated a method of releasing DNA at the point of measurement. Their findings are published in Small Methods.

Nanopores are very tiny holes that are found in biology or can be purpose engineered. There have been exciting advances in using nanopores as gateways that allow close monitoring as molecules pass through one by one. For example, the individual DNA bases passing through a pore have been identified allowing whole genome sequencing.

However, despite these remarkable steps in single molecule detection, it has been necessary to increase the concentration of DNA samples for successful measurement because there was no way of reliably getting the molecules to the measurement pore.

The researchers have created a 3D-integrated nanopore that can rupture cells immediately prior to measurement. The released molecules can be efficiently delivered to the sensing zone and measured without having to carry out any further steps that could introduce errors.

“Our sensor has two important parts. The first is a layer that contains numerous holes that are much smaller than a cell. An electrostatic field is used to rupture the cell and certain released substances can pass through holes while larger debris cannot, essentially providing a filter,” explains study first author Makusu Tsutsui. “Below this filter layer, separated by a spacer, is a single nanopore in a second membrane, where the measurements are made.”

When a voltage is applied, a current flows through the pore because of salt ions in the surrounding solution. This current is partially blocked when large DNA molecules are also passing through the pore, and the changes provide information about the large molecules. For example, whether the molecule—which can be millimeters in length—is folded.

“The filtering effect of our 3D-integrated nanopore prevents blockage of the measurement pore making it robust to use,” says study corresponding author Tomoji Kawai. “We therefore expect it to be used in new technologies for rapidly detecting mutant viruses at the genome level.”

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The article, “Detecting single molecule deoxyribonucleic acid in a cell using a three-dimensionally integrated nanopore,” was published in Small Methods at DOI: https://doi.org/10.1002/smtd.202100542

About Osaka University

Osaka University was founded in 1931 as one of the seven imperial universities of Japan and is now one of Japan's leading comprehensive universities with a broad disciplinary spectrum. This strength is coupled with a singular drive for innovation that extends throughout the scientific process, from fundamental research to the creation of applied technology with positive economic impacts. Its commitment to innovation has been recognized in Japan and around the world, being named Japan's most innovative university in 2015 (Reuters 2015 Top 100) and one of the most innovative institutions in the world in 2017 (Innovative Universities and the Nature Index Innovation 2017). Now, Osaka University is leveraging its role as a Designated National University Corporation selected by the Ministry of Education, Culture, Sports, Science and Technology to contribute to innovation for human welfare, sustainable development of society, and social transformation.

Website: https://resou.osaka-u.ac.jp/en