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Biological Nanopore Sensor Tech Shows Promise for Single-Molecule Detection of Proteins


NEW YORK – A team of researchers from Syracuse University has developed a new nanopore protein sensing technology that can detect a range of target analytes at the single-molecule level.

Described in Nature Communications last month, the technology, which combines a specific antibody-like binder with a common biological nanopore, allows scientists to measure various proteins without having to completely re-engineer the sensor.

"The detection occurs free in native conditions, and it's not restrained by the size of the protein," said Liviu Movileanu, a biomedical and chemical engineering professor at Syracuse University and the paper's corresponding author. "It's generalizable and can impact the field of molecular biomedical diagnostics."

According to Movileanu, one "persistent challenge" for the protein nanopore engineering field is that once the target analyte is changed, scientists typically have to redesign and re-optimize the nanopore sensor. "Proteins are quite diverse in Mother Nature," he noted. "Whenever we want to make some biosensor for a [different] protein-protein interaction or protein biomarker, you have to start the whole process again."

To solve this bottleneck, Movileanu's team devised a new class of nanopore sensors by combining two "key ingredients" — a protein nanopore and an antibody-mimetic binder.

The binder targets specific protein analytes and is fused to a monomeric beta-barrel protein nanopore from E. coli, tFhuA, via a flexible peptide tether. As the protein binder is positioned in proximity to the pore opening, the binding events outside the nanopore will change the current flowing through the pore. To target different analytes, researchers only need to change the binder that connects to the nanopore.

For their published study, the Syracuse researchers deployed the sensor technology for three different analytes: human small ubiquitin-related modifier 1 (hSUMO1), a model protein linked to various cellular processes including DNA repair and cell cycle; WD40 repeat protein 5 (WDR5), a chromatin-associated protein hub involved in epigenetic regulation; and epidermal growth factor receptor (EGFR), a prognostic biomarker in lung, colorectal, and breast cancers.

Overall, the study showed that the new approach can identify various target proteins with "high sensitivity and specificity" while maintaining the sensor’s architecture. "We identified the [binding] events with crystal-clear traces with no requirement for complex data algorithms," Movileanu noted.

The researchers also further challenged the method with 5 percent fetal bovine serum and found that the sensor could still report the presence of EGFR with single-molecule resolution.

"The big thing about the technique is, you can have the calibration curves and make it quantitative," Movileanu said. As such, with further development, the technology could potentially be used to detect protein biomarkers for clinical and diagnostics purposes, he added.

The technology is "really exciting," said Henry Brinkerhoff, a postdoctoral researcher at the University of Washington who has been studying nanopore protein sequencing. "Personally, I think it is the most promising way to do basic detection experiments with nanopores," he said.

According to Brinkerhoff, "one of the real draws" of nanopore technology is its arbitrarily scalable throughput, meaning that many pores can be combined to boost the detection. Therefore, the Syracuse researchers’ method can potentially lead to "a very engineerable and pretty modular system" for straightforward target detection, he said.

Despite its promises, Brinkerhoff also noted a few open questions about the new technology. For one, he said it remains unclear how much the system can ultimately be parallelized and what the upper limit for accommodating different probes is. "Can we truly make an array where we have 1,000 different recognizers and none of them get confused with each other?" he asked.

Another question is whether the sensors are specific enough to distinguish between a protein and the same protein with a slight modification, such as glycosylation.

"I think the approach developed in this paper is a really promising technique," said Jeff Nivala, a professor at the University of Washington whose lab has been developing nanopore proteomics technologies. "It is generalizable and seems to work with a wide range of different affinity reagents."

Since the technology deploys a so-called "bait and fish" strategy, it is conceptually different from the nanopore protein sequencing methods, which, analogous to nanopore DNA sequencing, pull a protein molecule through the pore to read the amino acid sequences.

Nivala said the technique described in the study could be particularly suited for biomarker detection, especially given its convenient readout.

However, he pointed out as a caveat that "you have to know what you're looking for." In addition, he said the approach requires highly specific affinity reagents that are compatible with the nanopore for different targets of interest, which can pose "another hurdle to jump through."

The study authors have filed a provisional patent pertaining to the method. While Movileanu currently has no plans for commercializing the technology, he said it could be licensed out to companies for commercial development.

Currently the sensor is able to capture analytes at nanomolar concentrations. Moving forward, Movileanu said one immediate goal for the team is to continue improving the sensitivity of the sensor, so it can detect analytes in the picomolar range with high precision.

"We're trying to raise the level of competition by going from the minor league to the major league," he said. "We expect to have a huge improvement in the detection level."