Salivating Over Spit
October / November 06
Volume 4 Number 5
At some point in the near future, it's possible that a dentist's order to spit may actually be a test for periodontal disease. The quick test in the dentist's chair may also indicate whether the patient has heart disease.
Researcher Anup K. Singh at the Livermore, Calif., campus of Sandia National Laboratories has led the development of the Integrated Microfluidic Platform for Oral Diagnostics, or IMPOD, which uses lab-on-a-chip technology to look at biomarkers in saliva to detect diseases or even bioterrorism agents. Unlike current blood-based tests that can take days, the IMPOD will yield results in a matter of minutes with a high degree of accuracy.
Singh brought to the project a background in chemical engineering, with his doctorate from North Carolina State University in 1995 after studying enzymatic and fluorescence immunoassays using unilamellar vesicles. He spent his first six years at Sandia working on biological sensing using microelectrical devices when in 2002 his project managers, Terry Michalske and Len Napolitano, pointed out a call for research proposals by the National Institute of Dental and Craniofacial Research arm of the National Institutes of Health. "I would have never looked at the NIDCR call myself," recalled Singh. "I didn't know spit about spit."
Nonetheless, in August of that year Singh soon found himself with a $4.2 million grant to develop an integrated microfluidic system for oral diagnostics in a four-year timeframe. And he also found himself deeply immersed in the biochemistry of spit.
"Saliva is a very complex fluid," said Singh. "It differs from person to person, and in each person with the time of day, in the presence of other disease factors and also with diet. That's not a problem with other fluids such as blood, which has a consistent composition."
What he and his team developed is a completely new way to analyze protein-based fluids. The IMPOD employs chip-based technology that has been revolutionizing analytical capabilities in arenas ranging from biomedical research to environmental remediation in recent years. For this application, a microfluidic chip is microfabricated to produce microchannels 20 to 40 microns deep, 100 microns wide and just a few centimeters long. The chip is coupled to microelectronics, optical elements, a fluid-handling system and analytical software in a hand-holdable package that in prototype form weighs less than five pounds. Using a photopolymerization process adapted from semiconductor manufacturing techniques to engineer cast-in-place nanoporous polymeric gels in the minute channels, the IMPOD is able to seamlessly integrate several sample manipulation steps to complete the saliva analysis. The gel polymerization is confined to microchannels that are exposed to light by means of photoinitiators, thus opening the door for customizable gel properties for different device applications.
To use the IMPOD, the clinician places tiny amounts of saliva in the IMPOD sample loading area with a syringe. A thin nanoporous membrane made by photopolymerization then captures the proteins in the sample, thereby concentrating it 100-fold. A sieving process purifies the proteins from smaller contaminants, and the proteins are rapidly mixed with a measured quantity of fluorescently labeled antibody probe that binds to disease biomarkers. The sample is then electrophorectically eluted off of the membrane and injected into a separation channel. Polyacrylamide gel electrophoresis separates the antibody-bound antigen and unbound antibody by size and charge. Laser-induced fluorescence then detects the unbound antibody and bound antibody peaks. Those peaks are compared with a calibration curve using samples with known amounts of analyte to estimate the concentration of biomarkers in the test sample. Singh estimated that the IMPOD would have a biomarker level of detection of 10-13 molar. The device would be capable of simultaneous multianalyte tests for up to eight analytes.
On commercialization, the IMPOD would be the first portable, point-of-care microfluidic device that could be easily operated in a clinician's lab. Less than 100 μL of saliva would be needed to run the automated test, which would take less than 10 minutes to complete. This differs from existing clinical immunoassays performed in microtiter plates in third-party labs that require large samples, expensive reagents, multiple steps of incubations and washing, and take hours to complete.
Singh and his research team have partnered with William V. Giannoble, professor of dentistry and director of the Michigan Center for Oral Health Research at the University of Michigan Clinical Center in Ann Arbor. Giannoble has assembled 100 human test subjects for the first longitudinal clinical trials of the IMPOD. Half of the study subjects have been diagnosed with periodontal disease through conventional test methods, and the others have been shown to be at low risk for disease. Over the next year, the subjects will be tested every two months, and will receive subtractive radiography testing at the six-month and one-year marks to measure any minute changes in bone loss.
"We will be testing the device's ability to be a diagnostic tool for the routine assessment of active periodontal disease, and may also be able to predict a patient's risk of future disease," said Giannoble. He noted enthusiasm for the possibility that a point-of-care device might soon be commercialized to detect not only periodontal disease but also other systemic diseases, and pointed to ongoing research showing that periodontal disease may be directly related to cardiovascular health. "Treatment of oral infection can reduce the risks for heart disease," he said.
Knowing that a patient already has or may be at risk for periodontal disease would help the dentist to determine the course of care, which could range from preventive measures such as antibiotics to interventions such as deep cleanings or surgery.
The IMPOD will also have to undergo vetting by the U.S. Food and Drug Administration before commercialization. The FDA will mandate whether saliva samples must be taken by a patient drooling into a cup, or if a clinician should collect saliva either by swabbing the oral cavity with a cotton applicator or collecting the gingival crevicular fluid from the tooth-gum interface. The University of Michigan tests will use samples collected on swabs using run-of-the-mill whole saliva from the oral cavity.
An analytical equipment manufacturer has expressed strong interest in bringing the IMPOD to market, and has estimated that it will need about two years to work up its design and manufacturing schemas. That timeframe dovetails with Singh's estimate that the FDA review process will take about two or three years, so both processes are expected to run in parallel.
Although the first commercial application of the device is expected to be for periodontal disease, variations could be used to detect other biomarkers in saliva, blood or urine.
"The technology we have developed is not limited to the detection of periodontal disease," Singh said, citing potential applications for lab-based protein analysis in research labs and pen-side veterinary diagnostics of diseases such as hoof-and-mouth disease.
Singh already has funding to look at the technology's potential to detect biological warfare agents in human blood or saliva. Currently, detecting the presence of bioagents relies heavily on environmental analysis technologies to determine if bioterrorism has taken place. "We have to complete the loop, and go beyond environmental detection to look at the people themselves," he said.
G. Jeffrey Hoch covers Sandia for Innovation.
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