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When will people be able to test blood glucose levels using just their saliva?

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Researchers at Brown University have developed a new biochip sensor that that can selectively measure glucose concentrations in a complex fluid like saliva. The new research is described in the cover article, "A 'plasmonic cuvette': Dye chemistry coupled to plasmonic interferometry for glucose sensing," appearing in the June 2014 issue of the journal Nanophotonics. After all, if you can test DNA and total genomes from a bit of saliva, why not also be able to test your blood glucose levels from your saliva without having to draw a blood sample in a test tube or stick your finger to get a drop of blood?

Their approach combines dye chemistry with plasmonic interferometry. A dependable glucose monitoring system that uses saliva rather than blood would be a significant improvement in managing diabetes. The advance is an important step toward a device that would enable people with diabetes to test their glucose levels without drawing blood.

The new chip makes use of a series of specific chemical reactions combined with plasmonic interferometry, a means of detecting chemical signature of compounds using light. The device is sensitive enough to detect differences in glucose concentrations that amount to just a few thousand molecules in the sampled volume.

“We have demonstrated the sensitivity needed to measure glucose concentrations typical in saliva, which are typically 100 times lower than in blood,” said Domenico Pacifici, according to the June 3, 2014 news release, "Progress on detecting glucose levels in saliva." Pacifici is an assistant professor of engineering at Brown, who led the research. “Now we are able to do this with extremely high specificity, which means that we can differentiate glucose from the background components of saliva.” For more information, see the website of Pacifici Research Group - Nanophotonics, the research group of Professor Domenico Pacifici.

The biochip is made from a one-inch-square piece of quartz coated with a thin layer of silver

Etched in the silver are thousands of nanoscale interferometers — tiny slits with a groove on each side. The grooves measure 200 nanometers wide, and the slit is 100 nanometers wide — about 1,000 times thinner than a human hair. When light is shined on the chip, the grooves cause a wave of free electrons in the silver — a surface plasmon polariton — to propagate toward the slit. Those waves interfere with light that passes through the slit. Sensitive detectors then measure the patterns of interference generated by the grooves and slits.

When a liquid is deposited on the chip, the light and the surface plasmon waves propagate through that liquid before they interfere with each other. That alters the interference patterns picked up by the detectors, depending on the chemical makeup of the liquid. By adjusting the distance between the grooves and the center slit, the interferometers can be calibrated to detect the signature of specific compounds or molecules, with high sensitivity in extremely small sample volumes.

Saliva is a complex solution

In a paper published in 2012, the Brown team showed that interferometers on a biochip could be used to detect glucose in water. However, selectively detecting glucose in a complex solution like human saliva was another matter.

“Saliva is about 99 percent water, but it’s the 1 percent that’s not water that presents problems,” Pacifici said. “There are enzymes, salts, and other components that may affect the response of the sensor. With this paper we solved the problem of specificity of our sensing scheme.”

Dealing with the 1 percent

The researchers knew that a plasmonic interferometer can detect glucose molecules in water. Detection of glucose in a complex fluid is more challenging. Controlling the distance between grooves and using dye chemistry on glucose molecules allows researchers to measure glucose levels despite the 1 percent of saliva that is not water.

They did that by using dye chemistry to create a trackable marker for glucose. The researchers added microfluidic channels to the chip to introduce two enzymes that react with glucose in a very specific way. The first enzyme, glucose oxidase, reacts with glucose to form a molecule of hydrogen peroxide.

Solving the problem of how the components in saliva are sensed

This molecule then reacts with the second enzyme, horseradish peroxidase, to generate a molecule called resorufin, which can absorb and emit red light, thus coloring the solution. The researchers could then tune the interferometers to look for the red resorufin molecules.

“The reaction happens in a one-to-one fashion: A molecule of glucose generates one molecule of resorufin,” Pacifici said, according to the news release. “So we can count the number of resorufin molecules in the solution, and infer the number of glucose molecules that were originally present in solution.”

The team tested its combination of dye chemistry and plasmonic interferometry by looking for glucose in artificial saliva, a mixture of water, salts and enzymes that resembles the real human saliva

The researchers found that they could detect resorufin in real time with great accuracy and specificity. They were able to detect changes in glucose concentration of 0.1 micromoles per liter — 10 times the sensitivity that can be achieved by interferometers alone.

The next step in the work, Pacifici says, is to start testing the method in real human saliva. Ultimately, the researchers hope they can develop a small, self-contained device that could give diabetics a noninvasive way to monitor their glucose levels.

There are other potential applications as well

“We are now calibrating this device for insulin,” Pacifici said, according to the news release. “But in principle we could properly modify this ‘plasmonic cuvette’ sensor for detection of any molecule of interest.”

It could be used to detect toxins in air or water or used in the lab to monitor chemical reactions as they occur at the sensor surface in real time, Pacifici said, according to the news release. The work is part of a collaboration between Pacifici’s group at Brown and the lab of his colleague Tayhas Palmore, professor of engineering. Graduate students Vince S. Siu, Jing Feng, and Patrick W. Flanigan are coauthors on the paper.

The work was supported by National Science Foundation (CBET-1159255, DMR-1203186 and HRD-0548311) and the Juvenile Diabetes Research Foundation (JDRF Grant 17-2013-483). For further information see, Nanophotonics. Volume 3, Issue 3, Pages 125–140, ISSN (Online), May 2014. The print edition is dated June 2014.

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