Molecular fishing

SYRACUSE, N.Y.—Thanks to a specific and customizable molecular “bait,” Dr. Liviu Movileanu, a professor of physics at Syracuse University, can find a single molecule in blood. Movileanu and recently graduated Ph.D. student Avinash Thakur presented their research, which could have wide-ranging applications from diagnostic tests to drug discovery, at the 64^th Annual Meeting of the Biophysical Society in San Diego in February.

 

In an article published in ACS Sensors and titled “Single-Molecule Protein Detection in a Biofluid Using a Quantitative Nanopore Sensor,” Thakur and Movileanu said: “Protein detection in complex biological fluids has wide ranging significance across proteomics and molecular medicine. Existing detectors cannot readily distinguish between specific and nonspecific interactions in a heterogeneous solution. Here, we show that this daunting shortcoming can be overcome by using a protein bait containing biological nanopore in mammalian serum.”

 

Movileanu said that he began “chasing protein‐protein interactions under disease‐like conditions” after his postdoctoral studies. He started to use nanopores, tiny holes in cell membranes through which scientists shoot electrical currents.

 

When an individual molecule, such as a protein, enters the pore, the electrical current changes in a way that enables scientists to identify the molecule’s identity. Movileanu knew that he needed to modify the system in order to understand how different proteins interacted with each other. One of the challenges was that grouped proteins are too large to fit into the nanopore, where the measurement typically takes place.

 

Thakur and Movileanu developed molecular “fishing” by fusing a modified receptor that acts like a “hook and bait,” via a short flexible protein “line,” to a protein nanopore “rod and reel.” They added an extra little protein that acts like a fishing “bobber.” When there is nothing on the “hook,” it bobs rapidly into and out of the nanopore. When something grabs hold, it stops moving around, alerting scientists that something is on the “hook.” A pore that is too small for big protein complexes acts as a sensor for protein interactions.

 

Movileanu explained that the development “is a 10-plus-year story with many detours and obstacles.” The key ingredient that made this happen is “the bobber,” he said.

 

Unlike fishing bait, where a worm might catch a trout or a catfish, Thakur and Movileanu’s bait is both extremely specific and totally customizable to find any protein of interest, and it even works in complex solutions like blood or biopsy samples. This precise protein engineering has practical significance in diagnostics, and because of its specificity there are no potential false‐positive signals produced by the constituents of a complex biofluid sample. Additionally, calculations with several of these “fishing rods” can reveal the concentration of the protein of interest in the solution.

 

As Movileanu said, “This technology uses ability to measure a tiny electrical current through an engineered protein nanopore. This approach is generic. It can be used for protein detection and biomarker discovery in complex biological fluids, such as blood, biopsies, cell lysates and solid tumors.”

 

He added, “This method can be integrated into nanofluidic devices—such as MinION DNA sequencer for long-read nucleic acid sequencing—that can host thousands of such sensors. They can potentially screen thousands of diverse proteins in a few runs.”

 

Such devices can screen targeted drugs against protein-protein interactions, specifically those involved in disease-like conditions such as those from cell signaling pathways under oncogenic conditions.

 

A non-provisional patent is now pending, and planning for licensing is facilitated by the Office of Technology Transfer at Syracuse University. Movileanu believes that this strategy “will certainly stimulate partnership with a major investor that will bring this prototype to next-generation molecular biomedical diagnostics.” He thinks that the target date for commercialization is two to four years.

 

According to Movileanu, “This sensor has realistic prospects in many biomedical areas. We demonstrated the proof of concept, and the next step is scaling it up to find the needles in a lot more haystacks. We anticipate a transformative impact of this technology in molecular diagnostics.”