Scientists Develop Sensitive Salivary Sensor

Issue date: 06.08.08

For people who dislike needles, medical tests that require a drop of saliva instead of a vial of blood will one day make a trip to a doctor or dentist much easier. But as scientists now construct the first of these saliva tests for early signs of cancer and other diseases, they continue to push the technological envelope in interesting ways.

A team of researchers supported by the National Institute of Dental and Craniofacial Research (NIDCR), part of the National Institutes of Health, report they have developed an ultra-sensitive optical protein sensor, a first for a salivary diagnostic test. The sensor can be integrated into a specially designed lab-on-a-chip, or microchip assay, and pre-programmed 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.

In their initial experiments, the scientists primed the optical protein sensor to detect the IL-8 protein, which at higher than normal concentration in saliva is linked to oral cancer. Using saliva samples from 20 people; half healthy, the others diagnosed with oral cancer — the sensor correctly distinguished in all cases between health and disease.

Importantly, the sensor achieved a limit of detection for IL-8 that is 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, or 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, Ph.D., a scientist at the University of California at Los Angeles and senior author on the above-mentioned paper in Biosensors and Bioelectronics, his group’s first step in widening the LOD window was to restructure the initiation of the fluorescent signal. They directly labelled the sensor-bound IL-8 with fluorescent probes, thereby cutting out the common intermediate step of using enzymes to amplify the signal. This streamlining improved the LOD of their saliva test to a level comparable with a standard ELISA blood test.

But Ho and colleagues decided to push the limit of detection even harder. Saliva naturally 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 six orders of magnitude less than one atom per cell.

To increase the sensitivity — and thus extend the lower limit of the LOD — Ho and colleagues 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. This creates the visual equivalent of static that heightens the intensity of the fluorescence and can lead to false positive diagnoses.

"When we talk about pushing a test’s limit of detection, or LOD, we’re referring to the signal to noise ratio," said Ho. "It’s really a matter of figuring out how to reduce the background noise and make the signal stand above the noise."

The National Institutes of Health