Scientists Develop Sensitive Salivary Sensor to Test for Early Signs of Disease

As scientists construct the first saliva tests for early signs of cancer and other diseases, they continue to push the technological envelope in interesting ways. As published in the August 2008 issue of the journal Biosensors and Bioelectronics, a team of researchers supported by the National Institute of Dental and Craniofacial Research (NIDCR) reports it has developed an ultrasensitive optical protein sensor, a first for a salivary diagnostic test. The sensor can be integrated into a specially designed microchip assay and preprogrammed to bind a specific protein of interest, generating a sustained fluorescent signal as the molecules attach. A microscope then reads the intensity of the fluorescent light—a measure of the protein’s cumulative concentration in the saliva sample—and scientists gauge whether it corresponds with levels linked to developing disease. The scientists primed the optical protein sensor to detect the IL-8 protein, which at higher than normal concentrations in saliva is linked to oral cancer. Using saliva samples from 20 people—half of them were healthy, the others were diagnosed with oral cancer—the sensor correctly distinguished between health and disease in all cases. Importantly, the sensor achieved a limit of detection for IL-8 roughly 100 times more sensitive than today’s blood-based Enzyme-Linked ImmunoSorbent Assay (ELISA) tests, the standard technique to measure protein in bodily fluid. The limit of detection (LOD) refers to a sensor’s ability to distinguish the lowest concentration of a protein or other target molecule apart from competing background signals.

According to Chih-Ming Ho, PhD, a scientist at the University of California at Los Angeles and senior au-thor of the above-mentioned paper, his group’s first step in widening the LOD window was to restructure the initiation of the fluorescent signal. They directly labeled the sensor-bound IL-8 with fluorescent probes, cutting out the common intermediate step of using enzymes to amplify the signal. This improved the LOD of their saliva test to a level comparable with a standard ELISA blood test. However, Dr. Ho and his colleagues decided to push the limit of detection even harder. Saliva contains much lower concentrations of protein than blood, and they wanted their sensor to attain the extremely high sensitivity that some future salivary diagnostic tests will likely require. Sensitivity refers to the smallest amount of a substance, such as a protein, that a diagnostic test can detect, which Ho said he hoped to extend down to the femtomolar range, or 6 orders of magnitude less than one atom per cell. To increase the sensitivity—and thus extend the lower limit of the LOD—Ho, et al sought novel ways to turn down the noise. Noise refers to the various ambient molecules in the saliva sample that typically stray to the sensor and bind, creating the visual equivalent of static, which can lead to false positive diagnoses. By utilizing a confocal microscope, they identified and focused on a location that has the maximal signal-to-noise ratio, extending the LOD by 2 orders of magnitude and pushing the envelope for the limit of detection. This will be an important capability in advancing salivary diagnostics.