Nano-sized silver cubes can make diagnostic tests easier to read

Engineers at Duke University have shown that nanosized silver cubes can be brighter by more than 150 times, making diagnostic tests that rely on fluorescence easier to read.

Combined with a new point-of-care diagnostic platform that has already been shown to detect small traces of viruses and other biomarkers, this approach makes such tests cheaper and more widespread. ..

Results published online May 6th in journal Nano character..

Plasmonics is a scientific field that traps the energy of a feedback loop called plasmon on the surface of a silver nanocube. When a fluorescent molecule is sandwiched between one of these nanocubes and a metal surface, the interaction between their electromagnetic fields causes the molecule to emit light more strongly.

Maiken Mikkelsen, James N. and Elizabeth H. Barton are associate professors of electrical and computer engineering at Duke, working with Duke’s laboratories to use plasmonics to develop new types of hyperspectral cameras and ultrafast optical signals. Has been created for nearly 10 years.

At the same time, Alan L. Kaganov, a professor of biomedical engineering at the Ashtosh Kirkoty Institute, said he was able to identify trace quantities of specific biomarkers from specific locations at a self-contained point of care diagnostic test. We are working on biological fluids such as blood.

However, the test relies on fluorescent markers to indicate the presence of biomarkers, so to see the faint light of a barely positive test requires expensive and bulky equipment.

Our research has already shown that plasmonics can increase the brightness of fluorescent molecules by tens of thousands or more. Using it to enhance a fluorescence limited diagnostic assay was clearly a very exciting idea. “

Maiken Mikkelsen, James N. and Elizabeth H. Barton Associate Professor of Electrical and Electronic Engineering, Duke University

“There are not many people who use plasmon-enhanced fluorescence for point-of-care diagnostics, some of which have not yet been implemented in clinical practice,” said Daria Semeniac, a graduate student at Chilcoty’s lab. I added. “It took about two years, but I think we have developed a functional system.”

In the new paper, researchers at the Chilkoti lab are building an ultrasensitive diagnostic platform called the D4 assay on a thin film of gold, the positively preferred anion of plasmonic silver nanocubes. The platform starts with a thin layer of polymer brush coating. This prevents researchers from sticking to the gold surface something they do not want to stick to it.

Next, researchers use an inkjet printer to attach two groups of molecules that are tailored to latch to the biomarker the test is trying to detect. One set is permanently attached to the gold surface and catches some of the biomarkers.

The other, when the test begins, is washed off the surface and adheres to another part of the biomarker, flashing a light, indicating that the target has been found.

After a few minutes before the reaction occurs, the rest of the sample is washed away, leaving only those molecules that have found a biomarker match, such as fluorescent beacons tethered to a golden floor.

“The real importance of the assay is the polymer brush coating,” Chilkoti said. “Polymer brushes allow us to store all the tools we need for a tip while maintaining a simple design.”

The D4 assay is very good at capturing small traces of certain biomarkers, but when only traces are present, fluorescent beacons can be difficult to see. The challenge for Mikkelsen and her colleagues was to place a plasmonic silver nanocube on top of the beacon to overcharge the beacon’s fluorescence.

But, as is usually the case, this was easier than it sounds.

“The distance between the silver nanocube and the gold film determines how bright the fluorescent molecule will be,” said Daniela Cruz, a graduate student working in the Mikkelsen lab. “Our challenge is to make the polymer brush coating thick enough to capture the biomarkers (and only the biomarkers of interest), but not enough to enhance the diagnostic light. It was to thin it. “

Researchers have tried two approaches to solve this Goldilocks mystery. They first added an electrostatic layer that bound to the detector molecule that carried the fluorescent protein, creating a “second floor” over which the silver nanocubes could be placed. They also sought to functionalize the silver nanocubes to attach directly to each detector molecule, one-to-one.

Both approaches succeeded in increasing the amount of light from the beacon, but the former showed the best improvement, increasing its fluorescence by more than 150 times.

However, this method also requires the additional step of creating a “second floor”. This adds another hurdle for designing how to do this task in the laboratory as well as in commercial point-of-care diagnostics. Also, the second approach did not significantly improve fluorescence, but it did improve the accuracy of the test.

“Building a microfluidic lab on-chip device with either approach takes time and resources, but both are theoretically viable,” says Casio Fontes, a graduate student at the Chircoti Institute. Says. “That’s the direction of the D4 assay.”

And the project is moving forward. Earlier this year, researchers used the preliminary results of this study to win the $ 3.4 million R01 Research Award for five years from the National Heart, Lung, and Blood Institutes.

Collaborators have optimized these fluorescence enhancements, while enabling wells, microfluidic channels, and other low-cost solutions to perform all these steps automatically, making low-cost common smartphones a reality. We plan to integrate it into a camera-readable single-step diagnostic device. Cost device.

“One of the major challenges of point-of-care testing is the ability to read results that typically require very expensive detectors,” Mikkelsen said. “This is a major obstacle to disposable testing that allows patients to monitor chronic illness for use at home or in low resource settings. This technology is not only a way to avoid bottlenecks, Accuracy and thresholds of these diagnostic devices. “