The use of nanopores to detect potential diseases

forschung leben – the Magazine of the University of Stuttgart (April 2023)

Two working groups at the University of Stuttgart are part of the nanodiag BW future cluster. Research is being carried out to expand medicine through prognostics in order to detect the development of diseases at an early stage. The basis for this is a new type of bioanalysis, the analysis of biomolecules using nanopores.
[Photo: Hahn-Schickard]

A new type of bioanalysis could be used to expand the field of prognostics but this would requires a much deeper fundamental understanding of molecular processes. The so-called Cluster4future nanodiag BW (Next generation innovation network Nanodiag BW), which includes two working groups from the University of Stuttgart among others, has set itself the goal of achieving this.

The human body is made up of 100 trillion cells, each of which performs a specific task within the body. Because cells have a limited life span, they divide on a regular basis in a highly complex process that produces incredibly accurate copies of the parent cell. "However," as Prof. Stephan Nußberger explains, "medical scientists now also know that even our lifestyles can cause minimal changes in a gene or in the formation of new proteins within a cell." This can lead to uninhibited cell proliferation and potentially to poorer resistance to pathogens. "They can also cause diabetes, cancer, or infectious diseases," says the biophysicist, who conducts his research at the University of Stuttgart's Institute of Biomaterials and Biomolecular Systems. "Whilst diagnosing such diseases is often straightforward," says Nußberger, "medical scientists are hoping to augment diagnostics with prognostics going forward." This, he explains, would make it possible to detect the problem at an early stage and to initiate a less aggressive treatment to counteract it in good time.

Whilst diagnosing such diseases is often straightforward, medical scientists are hoping to augment diagnostics with prognostics going forward.“

Prof. Stephan Nußberger

Cluster for a new kind of bio-analytics

Achieving this is the goal that Cluster4future nanodiag BW (Next generation innovation network nanodiag BW) has set itself. The project group includes the Hahn-Schickard-Gesellschaft für angewandte Forschung (Hahn-Schickard Society for Applied Research), which is closely associated with the University of Stuttgart, seven other universities and several non-university-affiliated research institutes, as well as nine commercial enterprises. The University of Stuttgart is represented by Nußberger's research group and a group led by Prof. Christian Holm of the Institute for Computational Physics. The specific aim is to develop a new type of bioanalysis involving the use of nanopores to analyze biomolecules.

This diagram shows how the electrical conductivity of a nanopore temporarily decreases as a protein fragment passes through the pore.

The process exploits the way cells work. "All cell walls include pores through which metabolism takes place," Nußberger explains. These enable the passage of things such as ions, sugar molecules, and biomolecules. "Pores have diameters ranging from 0.1 to five nanometers, depending on what they transport." 0.1 nanometer is equal to one atomic diameter and one nanometer is one millionths of a millimeter.

The engineered replication of this transport mechanism can be used to detect single molecules: an electrically conductive liquid chamber is separated into two areas by a porous membrane. The molecules to be analyzed are in the liquid, which is usually a saline solution. Applying an electrical voltage causes a current to flow through the porous membrane, because the ions in the liquid migrate, whereas the current drops if a molecule gets trapped in the pore or passes through it. The specific characteristics of the change will indicate what exactly has blocked or passed through the pore.

The pore being studied is divided into millions of micro droplets in a process known as "emulsion coupling".

Measurements in the millisecond range

"So far," says Christian Holm, "nanopore technology has only been used commercially for the analysis of DNA. But what our research is about is the characterization and even the direct sequencing of proteins." Sequencing, in this context, means determining the sequence of amino acids that make up a protein. Initially the project team will be studying bionanopores, which are specially produced biological pores. The molecules that will be analyzed will only differ slightly from one another, for example, by a handful of atoms at a specific location – analogous to the aforementioned lifestyle-related changes, to which end the project partners will need to be able to measure minuscule current differences in the millisecond range. The currents in question might differ by one trillionth of an ampere. To put this in perspective the current that flows when charging a smartphone is about one ampere.

"For us to succeed in all of this," Holm explains, "we need to have a comprehensive understanding of nanopores and how they interact with proteins." And comprehensive in this case really does mean comprehensive: the relevant events take place in just a few cubic nanometers, although tens of thousands of individual particles are involved in this process at the atomic and molecular level, some from the aqueous solution, some from the protein to be analyzed, and some from the pore surface. "We still don't understand the details of the processes involved," says theoretical physicist Holm, which is where his working group comes in.

The so-called PICO method uses two distinct, DNA-labeled antibodies that target specific proteins, shown here in blue and yellow.

"We model the proteins, the pore, and the fluid on the computer," he explains. The model reflects such things as which parts of a protein are more rigid or more flexible, and how strongly its atoms interact with each other as well as the relevant mechanical and electrical forces. "Then we use the model to simulate the passage of proteins through the nanopore to see how the proteins move within it and which interaction we would expect to produce which electrical current signal."

The cluster members continuously compare the results from these simulations with those from experiments being carried out in Freiburg. "The aim then is to collaborate with a team at RWTH Aachen University to develop a program that can reliably analyze real-world electrical current signals," says Holm. Artificial intelligence will be used to perform the analysis, because the signals will be noisy and will often result from protein changes that have never been empirically measured before, because of the huge number of variants.

For us to succeed in all of this, we need to have a comprehensive understanding of nanopores and how they interact with proteins."

Prof. Christian Holm

Nußberger's team's task will be to provide control measurements to help develop the electrical current measurement technology. “We already have an optical system that can detect whether a pore is currently open or closed,” says Nußberger. “But when it comes to the production of bionanopores, we still can't check whether a single pore is created in the relevant area of a membrane, which is what we want, or whether several pores are created, which means that measuring just the current alone could falsify the result.”

From basic research to product development

The cluster members are then planning to extend the process to solid-state nanopores in a follow-up project. Nanopores, as Holm explains, "are pores in artificially produced thin membranes made of substances such as graphene." The pores would be only one atomic layer thick, because graphene is a two-dimensional material. "This," as Holm explains, "would mean that there could really only be exactly one protein building block migrating through a pore at any given time, which would enhance the signal resolution.” But, up to now, this is only a theoretical possibility, because we cannot yet produce these pores in a sufficiently reproducible manner. It would also be more of a challenge to measure the signal because it would be even weaker and shorter than that of bio-nanopores and there would be more background “noise”. But, on the other hand, solid-state nanopores would be more robust and would lend themselves to a greater range of uses and could be integrated into electronic systems. It would also be possible to place a large number of solid- state nanopores next to one another on a membrane, which would enable the measurements to be carried out in parallel: the optimal analyzer.

The cluster will operate for three three-year periods and will receive 45 million euros of funding from the German Federal Ministry of Education and Research. "What we want to do in the first three years," says Holm, "is to lay the development foundations after which we'll turn our attention to the most promising approaches in the following three years, before devoting the final three years to creating the product."

Prof. Dr. Stephan Nussberger, E-mail, Phone: +49 711 685 65002
Prof. Dr. Christian Holm, Email, Phone: +49 711 685 63701 

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