Finding and identifying an infection is a tricky challenge on the best of days for doctors and hospitals, and rapid identification of the guilty bacterial or viral agents can be the difference between life and death.
Now a team from Stevens Institute of Technology, in partnership with one of the region's top-ranked medical centers, is working to refine a new way to do it more quickly and accurately.
"The current technology takes anywhere from one to three days to identify an infection," explains materials science Professor and Associate Dean Matt Libera, principal investigator on the project. "That is a long time for a doctor to not know whether or not an infection is present, or to wonder about prescribing the right antibiotic, or to overprescribe for a condition that antibiotics will not be able to treat anyway.
"We think we can help improve this process dramatically."
Microgel tethering DNA probes
The new detection technology is based on gel-tethered DNA and RNA detection probes, an advance co-invented by Libera, former Stevens Ph.D. student David Dai and Dr. Salvatore Marras at the Public Health Research Institute in Newark, New Jersey.
It works like this: RNA is extracted from drops of blood sampled from a patient, and the extracted RNA solution is sprinkled onto a glass microscope slide arrayed with tiny dots of specially designed, formulated and patterned microgel.
Within each spot of gel, tiny bits of DNA known as molecular beacons — each acting as tags or probes for a specific bacterium or virus — touch the RNA solution. If the molecular beacons meet a matching piece of RNA, they glow fluorescently; the glow can be seen using a simple microscope. (Sometimes RNA from the blood sample is first amplified, or copied many times, to improve the detection process.)
While similar techniques have been around for a while, explains Libera, they haven’t worked well, chiefly because molecular beacon DNA probes don’t function optimally when attached directly to a microscope slide.
"DNA probes don't work as well as we would like when they are attached directly to solid surfaces," says Libera.
To get around this slippery issue, he and his students have developed and refined new types of microgels, manufactured and patterned in arrays using electron beams, that share some of the properties of both liquids and solids.
"There are lots of different hydrogel systems out there, but our gels are unique. Each one is like a microscopic piece of cotton candy," he explains. "The microgel center, like the cardboard handle in the cotton candy, is pretty firm and dense. But the microgel edges are wispy and soft like the cotton candy itself. So the DNA probes we use get tethered to the softest, most liquid-like edge of the microgels, where they work most effectively."
Total time for a test result, if the system turns out to work, will shorten dramatically.
"We’ve measured detection events in spans of time as short as 20 to 30 minutes," says Libera. "This approach can be quick and very sensitive, down to as few as five bacterial cells in a milliliter of cultured blood."
Now the technology is heading toward a potential clinical application.
"Early on, a venture capital guy in New York told us that this technology wouldn’t go anywhere if we couldn’t convince clinicians that it can make a difference," recalls Libera. "So we began working with Hackensack Meridian Health's Hackensack University Medical Center."
Key collaborators in the medical center's Department of Pathology include Dr. Gary Munk, Clinical Director of Clinical Virology, and Dr. Tao Hong, Chief of Microbiology and Molecular Diagnostics.
"I look at this project as a perfect match," says Hong. "Stevens has a great idea and two exceptionally talented materials students working here, and we have the laboratory facilities, the pathogen samples and the experience as they attempt to improve upon the current best technology, which takes about one hour to identify what an infection probably is."
"This is what discovery is about," adds Munk. "Scientific curiosity, the eureka moment. We are working together with Stevens because they bring that spark, that curiosity to the lab environment and this important challenge of identifying infections as quickly as possible. Collaboration is important."
Students contribute key insights
In Hackensack University Medical Center labs, Stevens Ph.D. candidates Youlong Ma and Feiyue Teng work closely with Drs. Munk and Hong, assisting in the process of testing various gels with blood samples extracted from infected patients.
"Our probes perform five to six times better than the traditional method of using microarrays on a glass slide," says Ma. "Our approach is quick and it's sensitive, and the entire detection system can be made very small."
Now Teng is working on a newer companion project to try to identify viruses such as H1N1, H3N5 and other flu strains.
"There are more than 100 subtypes of viruses, so quick detection is very important, before the patient even shows symptoms," she says. "The doctors at Hackensack University Medical Center feel that our approach using NASBA — nucleic acid sequence-based amplification — of viral mRNA may be clinically very valuable as a way to quickly amplify and detect viruses in the hospital environment."
Undergraduate Joshua Ross '18, a chemical biology major, has also made key contributions as a Stevens CHI (Center for Healthcare Innovation) Scholar. Ross worked on using orthogonal chemistries to develop a new type of microgel that will allow the tethering of many different biomolecule probes to the microgels.
What's next? The Stevens-Hackensack team is already close to making a prototype quick-test kit to detect for a bacterial infection, and the same approach may well be extended to viruses and other pathogens.
"A lot of arrows are all pointing in a really positive direction right now," concludes Libera. "These arrows may lead, for example, to a small portable device connected to a cell phone that can rapidly test for a large panel of different possible pathogens.
"We have more to do, and no doubt we will experience unforeseen challenges. That’s the nature of R&D. Still, within a year or so, we are hoping to have a prototype test that can be used experimentally in the hospital environments and validated using other FDA-approved testing methods."