University of Freiburg: Using Nanopores to Discover Factors That Influence Illness

The physiologist Prof. Jan Behrends from the University of Freiburg is the deputy spokesperson for the network “nanodiag BW” that brings together fundamental science, applied research, and industry in the field of nanopore technology. The researchers believe this technology has an enormous potential for medical diagnostics, which could have an impact on the prevention and treatment of illnesses like Alzheimer’s and cancer. The project has already reached the final round of the “Clusters4Future” competition, which only 15 out of 117 candidates managed.

The small silver-gray box in Jan Behrends’ hand seems unspectacular: It has a hatch to open, two buttons, and a blue display. Yet this small box is an analytical instrument with astonishing capabilities. It can detect and distinguish between individual molecules with the help of so-called nanopores. In this process, the structure of the analyzed molecules and physicochemical properties are determined.

Jan Behrends is Professor of Physiology at the University of Freiburg. He not only helped develop this small box, which is being realized by a company that he founded; his research group in Freiburg also works on the fundamental aspects of this new technology. Behrends is also the deputy spokesperson of the Zukunftscluster-Initiative (Future Cluster Initiative) “nanodiag BW” which brings together actors from fundamental science, applied research, and industry and which wants to turn Baden-Württemberg into a future center of nanopore technology. The first hurdle has already been cleared: “nanodiag BW” is one of the 15 finalists in the “Clusters4Future” competition of the German Federal Ministry of Education and Research. An independent jury of experts selected the project from Freiburg out of 117 candidates, and the nine-month conception and planning phase began in October 2021.

Nanopores made by bacteria

“Nanopores are the umbrella term for all kinds of holes on a molecular scale in an electrically insulating material,” explains Behrends, adding: “This can be a very thin layer of a mineral material or a biological membrane.” Biological nanopores consist of proteins and, for example, form channels through which the exchange of substances between the inside of the cell and the external environment occurs. “We use artificial cell membranes that we produce ourselves in our experiments,” says Behrends, adding: “Into these, we integrate individual nanopores produced by bacteria.” For example, the scientists are using the poison aerolysin, which forms pores and is produced by bacteria of the aeromonas hydrophila species. Alternatively, pores on a nanoscale can be drilled into thin layers of silicon nitride with an electron beam. “Bacteria can make more precise holes than we can,” says Behrends. That is why bacterial pores are a specialty of the researchers from Freiburg, because it is important that the pores always have a precise and identical form in order to achieve precise measurement results in the molecular analysis.

The process works roughly like this: The artificial cell membrane with a nanopore “swims in a conducting medium,” says Behrends, for example, a saline solution to which voltage is applied. Current flows through the pore as a result. “Say a non-conducting substance, like a peptide, enters the pore, for example because of diffusion or because it has a charge itself, we can measure the blockage of the current,” says Behrends. In other words: When biomolecules travel through the pore, the electrical conductivity of the pore changes in a characteristic way. The type of change tells us what kind of molecule it is and what kind of form and sequence it has.

Helping to advance the research of Alzheimer’s, cancer, and viruses

The research group from Freiburg focuses primarily on the analysis of amino acids and peptides. “Thanks to the aerolysin pore, we hope to be able to distinguish between peptides with great precision, even if they have the same mass but a different spatial structure,” says Behrends. This differentiation between isomers, which is extremely complex and requires a huge effort in traditional mass spectrometry, could provide an insight into the chemical modifications of protein sequences, for example.

We know that the so-called post-translational modifications play a role in illnesses like Alzheimer’s or cancer, and researchers hope to gain new knowledge in virology as well. They therefore believe that their technology has a great potential for medical diagnostics. Especially in the area of epigenetics, nanopore technology could help form a better understanding of processes on a cellular level and thus provide more precisely fitting ideas for the prevention and treatment of illnesses, according Behrends, who adds: “We want to develop a diagnostic platform for all of this in our cluster.”

Making Baden-Württemberg a site of nanopore technology

So far, this technology is “represented only sparsely in Germany,” which is why the future cluster finalist now plans to establish it in Baden-Württemberg and to “develop a critical mass” – including a graduate funding program. The project is coordinated by the Hahn-Schickard-Gesellschaft for applied studies in Freiburg, the director of which is Felix von Stetten who is also the head spokesperson of the future cluster finalist. Other institutions involved are the University Medical Center Freiburg, the Max Planck Institute for Immunobiology and Epigenetics, the Fraunhofer Institute for the Mechanics of Materials, as well as several start-ups from Freiburg. Other partners are the University of Stuttgart and representatives from the Innovation Alliance Baden-Württemberg, including the Natural and Medical Science Institute in Reutlingen, the Institute for Laser Technology and Medicine in Ulm, and the Hahn Schickard Institute in Villingen-Schwennigen. Larger companies, like Bosch and Endress & Hauser, are also involved. The project is based on a previous collaboration between several actors in the field of nanopore analysis in Baden-Württemberg, for which the state government granted seed capital of roughly five million euros over two years in 2020. With this funding, they are also working on further key areas of “nanodiag BW”: namely, the training of artificially produced pores to have the same precision and reproducibility as the bacterial pores, as well as integrating nanopores directly into electronic microchips.

Funding will be decided in July 2022

In the final round of the competition, which began in October 2021, the future cluster finalist now has the chance to develop their ideas further. The final decision will be made in July 2022. “The project wants to develop applications,” says Behrends, adding: “We also have to raise funds from industry.” Should the future cluster finalist be successful, funding could be made available for three times three years – with a maximum funding volume of 15 million euros for each three-year cycle.

Should this succeed, many more of these boxes will turn up in the labs of universities, research institutes, biotech companies, and pharmaceutical companies that could standardize and simplify the use of nanopore technology, thus potentially offering a much broader range of applications in the future.

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