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Chemical and biomolecular engineers at Georgia Tech have developed a plug-and-play platform that's simple, flexible, and easy to use without costly lab equipment, for detecting protein biomarkers of disease.

Their work could unlock a new wave of at-home testing options and provide new diagnostic capabilities in parts of the world where medical resources are scarce.

The testing platform fills a gap in using cell-free synthetic biology for disease detection. Existing cell-free tools have proven effective at measuring DNA, RNA, and other small molecules, but not proteins. That's an important advance because proteins in viruses or bacteria tend to change less than the DNA or RNA sequences that encode those proteins. They're also easier to detect since they can be found on the outside of cell walls or free-floating in biofluids.

"Diagnosing disease and democratizing medical care by putting it into the public's hands has great potential. You can have a big impact on a lot of people," said Mark Styczynski, William R. McLain Endowed Professor in the School of Chemical and Biomolecular Engineering.

"I think about that a lot in terms of the developing world, but also there's a lot of health care inequality even in the United States. Studies have shown your ZIP code can determine your life expectancy. You can think about people in sub-Saharan Africa or people in rural Appalachia all benefiting. They're among those who need more access to low-cost tools."

Styczynski and a group of researchers led by former Ph.D. student Megan McSweeney have their test in the journal Science Advances.

They described it as a modular cell-free protein biosensor platform. Cell-free means the team uses the kind of machinery found in cells but engineers it in the lab rather than in cells; and modular because they showed their platform can easily be adapted to detect a variety of proteins.

The tool can produce a visual result akin to a swimming pool pH test strip, or it can provide more precise information for use in a clinical or research lab.

"Flexibility was one of the things we wanted to focus on most, because we know just how powerful that feature is for synthetic biologists and engineers making these biosensors, and specifically for proteins," said McSweeney, first author of the study and now a postdoctoral scholar at Stanford University. "The level of flexibility that we achieved here is quite astounding."

In their paper, McSweeney and the researchers successfully detected a biomarker for malnutrition and a protein from SARS-CoV-2, the virus that causes COVID-19. The proteins they targeted in those two cases were vastly different in size, demonstrating a wide range of potential uses.

Experiments showed the test worked in pooled samples of human blood serum and saliva. McSweeney said they expect that the sensing platform would work across many other biofluids.

"It can take years of development and potentially millions of dollars to make one lateral flow test, which is what your at-home COVID test is," Styczynski said. That's why their platform is so powerful: changing the target is as simple as changing a few of the ingredients, without needing to optimize the testing protocol or reengineer all the parts of the test.

"We can easily swap these out, and then we don't have to worry about all that other crazy, multimillion-dollar development. It just works," he said.

How? The researchers use RNA polymerases, which can turn genes on and off. They attach two pieces of those to what are essentially very simple antibodies. Those antibodies are designed to find and bind with a particular protein. When they do, the RNA polymerase pieces that are now next to each other will connect.

When they "click" together, the RNA polymerase activates an enzyme that causes a dye molecule to change color. In the simplest version of the test, finding the target protein results in a range of colors—from yellow to orange to red to deep purple—depending on how much protein is present.

For a lab version of the test, the results could be a fluorescent output that could be plugged into lab equipment for more detailed analysis.

"We wanted to also frame this technology as something that's versatile enough to be used by people who have advanced lab equipment and resources," McSweeney said. "Our platform is flexible enough, I think, to cater to those people's needs, potentially increasing their throughput, their consistency, and their quality of data."

The test's modularity opens the possibility of use in disaster, first-response, and military settings, too. Styczynski suggested the platform could become a field kit for those scenarios, where technicians with a bit of training could easily mix the elements of the test on the fly to detect a variety of pathogens or biomarkers. The kit would have a number of raw materials and a set of "recipes."

Along with former Georgia Tech Ph.D. student Monica McNerney, McSweeney and Styczynski have applied for a patent on their approach. The research team also included Ph.D. students Alexandra Patterson and Kathryn Loeffler, undergrad researcher Regina Cuella Lelo de Larrea, and Garry Betty/V Foundation Chair and Professor Ravi Kane.

The researchers continue to develop the tool. They're working to detect even lower levels of target proteins and make the test platform more user-friendly. For example, their preliminary work showed it can withstand the process of lyophilization, which eliminates the need for special storage or refrigeration. But they said more work is needed to ensure the test would be shelf-stable in longer-term, real-world conditions.