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Potential Diagnostic Metabolites Detected With Nanopore Method

NEW YORK (GenomeWeb) – A team from the University of Groningen has demonstrated that it is possible to directly detect small molecule metabolites using a sample preparation-free single-molecule nanopore method.

In work supported in part by Oxford Nanopore Technologies, the researchers established an approach for finding and quantifying levels of glucose or an amino acid called asparagine with a cytolysin A (ClyA) nanopore paired with protein sensors that are based on adapted versions of bacterial binding proteins that specifically interact with these molecules.

The team's results, published online today in Nature Communications, suggest that the nanopores produced characteristic electronic signals when the target molecules moved through the pore — an approach that proved feasible for several different sample types considered for the paper.

"[U]sing proteins from a protein family that in cells recognize an enormous variety of molecules, we show that ClyA nanopores can report the concentration of glucose and asparagine directly from samples of blood, sweat, and other bodily fluids," corresponding author Giovanni Maglia, a chemical biology researcher at the University of Groningen, and his co-authors wrote.

"Incorporation of the nanopore into portable electronic devices will allow developing sensitive, continuous, and non-invasive sensors for metabolites for point-of-care and home diagnostics," the authors proposed.

While electronic methods for detecting metabolites have been devised in the past, the team noted, these strategies have been limited by their reliance on enzymes that oxidize a target molecule to produce a signal. That approach is primarily used for at-home or implantable glucose sensors, though problems remain for that application as well.

In an effort to expand the repertoire of small molecules that can be detected electronically, the researchers worked on a ClyA nanopore-binding protein system that might eventually make its way into such a device.

Generally speaking, they explained, the electrical signal produced when ions move through a nanopore in response to an applied electrical potential can provides insights into the types of molecules moving through a pore.

When it came to adapting this system to detect glucose, the team employed a glucose-binding protein that is trapped in the pore for a set amount of time under specific electronic and osmotic conditions.

The researchers demonstrated that the profiles produced as current moves through the pores shifted depending on the glucose-binding protein's open or closed configuration, which was in turn influenced by the presence of glucose on one side of the nanopore. They went on to show that this approach could be applied to nanoliter- or microliter-scale samples of blood, sweat, urine, and saliva.

From there, the team also adapted the approach — an amino acid that may act as a marker for post-stroke brain damage or Parkinson's disease — with another binding protein to detect asparagine. It also explored the possibility of measuring glucose and asparagine in the same sweat sample, showing that the nanopore blockades produced by each binding protein could be distinguished from one another.

The team is continuing to search for binding proteins that can be used in the nanopore system, hoping to come up with similar strategies to detect still more substrates in bodily fluids and other sample types. In a statement, Maglia noted that such proteins "need to be tuned to work with the pore."

"[A]t the moment, we don't really understand the mechanism for this, so finding the right proteins is a matter of trial and error," he explained. Even so, Maglia suggested, "If we can create a system with proteins that are specific to hundreds of different metabolites, we will have created a truly disruptive new technology for medical diagnostics."