Researchers supported by the National Institute of Dental and Craniofacial Research (NIDCR), part of the National Institutes of Health, have engineered a portable, phone-sized test that in minutes measures proteins in saliva that may indicate a developing disease in the mouth or possibly elsewhere in the body.

The point-of-care test, one of several saliva-based diagnostic devices now under development with NIDCR support, one day in the future could become a common sight in the dentist’s office. As envisioned by the researchers, a dentist would collect a small saliva sample with a patient’s consent, load it into the diagnostic cartridge, start the assay, and have a read out waiting after a cleaning or a dental procedure has been completed.

Called IMPOD, the device is described in the March 27 issue of the Proceedings of the National Academy of Sciences. In the report, the scientists offer the results of proof of principle experiments in which IMPOD reliably measured the concentrations of MMP-8, an enzyme associated with chronic inflammation of the gums called periodontitis.

“The IMPOD is designed to measure up to 20 analytes, or biochemicals, at once,” said Dr. Anup Singh, a chemical engineer at the Sandia National Laboratories in Livermore, Cal. and senior author on the paper. “We haven’t scaled up to that point, but we are doing multi-analyte analyses in the laboratory. Our greatest need right now is validated biomarkers to enable further clinical studies. The basic engineering of the device has been completed.”

According to Singh, he and his colleagues were intrigued a few years ago by the many potential advantages of saliva as a diagnostic fluid. These include easy collection, no painful needle sticks, portability of the tests, and potentially a lower cost to patients than blood assays.

But they were initially daunted by the research task at hand. “Saliva is a mirror of blood, but with a caveat, said Singh. “It’s not an exact mirror, meaning everything that is present in blood is present in saliva but at concentrations 1,000 to 10,000 times lower. It’s diluted by saliva and the other secretions in the mouth. So we needed sensitivity 1,000 to 10,000 times better than we’d need if we were screening serum samples.” Sensitivity refers to the lowest amount of an analyte that a test can detect.

Singh said he and his colleagues chose to use a lab-on-a-chip device. The term refers in this case to a microchip, roughly the size of a laboratory slide, containing networks of tiny channels and chambers in which the salivary assay, or biochemical test, is performed. For the researchers, the challenge was to design the entire assay to fit into the microchip. The scientists also had to miniaturize the components required to run the microchip, such as the power supply and optical detection unit, and integrate them into a rapid, fully automated diagnostic system.

The result is the IMPOD, short for Integrated Microfluidic Platform for Oral Diagnostics. The process begins with a series of microwells, each as distinct as fingers on a hand. One well holds the saliva sample, while the other wells contain cleansing buffering solution and antibodies that are preprogrammed to bind the specific protein of interest in saliva. The antibodies are tagged with a fluorescent dye that can be illuminated and measured at the end of the assay.

With the punch of a button, the contents of the wells are released and merge into a single channel about 40 microns wide, or roughly the width of a human hair. As the mixture flows in these tight quarters, the antibodies readily find the proteins of interest, tag them, and continue forward to be trapped on a porous gel membrane that serves as a filter.

“The bound proteins stack against the membrane because they are too big to squeeze through the pores,” said Dr. Amy Herr, also at Sandia National Laboratories and lead author on the paper. “Conversely, the many smaller molecules in saliva flow right through the pores and are filtered out and routed to a waste chamber.”

Thereafter, a voltage reversal releases the trapped proteins from the gel. They continue down a channel, where a standard diode laser shines onto the fluorescent tags and quantifies the concentration of the protein in the sample. The dentist reads the result and determines whether the protein levels correlate with a given disease.

To put their lab on a chip to the test, they collected saliva from 23 people — 14 with periodontitis and nine in good oral health. Loading roughly one-tenth of a drop of saliva for analysis, the IMPOD processed the samples and produced a result in less than five minutes. The results showed that on average those in good oral health had lower concentrations of MMP, while people with periodontitis on average had elevated levels of the tissue-damaging enzyme. The results were confirmed with a standard ELISA blood test, currently the gold standard.

“The data correlate nicely with those from other types of studies that show that MMP-8 is indicative of periodontitis,” said Dr. William Giannobile, a researcher at the University of Michigan School of Dentistry in Ann Arbor. “There is every reason to believe additional biomarkers for periodontitis can be identified, as the work to catalogue salivary genes and proteins nears completion and our understanding of periodontal disease continues to advance.”

The researchers noted that IMPOD, although developed for saliva, could be used to test other diagnostic fluids such as blood and urine.

The National Institute of Dental and Craniofacial Research is the Nation’s leading funder of research on oral, dental, and craniofacial health.

The National Institutes of Health (NIH) — The Nation's Medical Research Agency — includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. It is the primary federal agency for conducting and supporting basic, clinical and translational medical research, and it investigates the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit www.nih.gov.