|Scientists Develop Sensitive Salivary Sensor
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
As published in the August issue of the journal Biosensors
and Bioelectronics, 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 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
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
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 labeled 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."
Leyla Sabet, a member of Dr. Ho’s lab and a lead author on the
paper, explained that the group already filtered out other wavelengths
of light that might pollute the signal. That left them to parse
the fundamental — and often overlooked — subject of
where to collect the light. Does the signal-to-noise ratio vary
within or above the fluorescent pathway of light? If so, is there
a precise spot that offers the highest signal and the least noise?
But to answer the where question, the group first needed a better
optical tool to collect the light and see what’s there. They utilized
a confocal microscope, which gathers all of the fluorescence and
has the added advantage of locking onto a single slice, or horizontal
plane, of light and then viewing it from multiple points.
Sabet and colleagues began by locking the focus of their confocal
microscope on the surface of the sample, where signal and noise
typically coalesce. From there, they moved up from the surface
one micron at a time, collecting the light and calculating the
signal to noise ratio at each point.
"We identified a location that has the maximal signal-to-noise
ratio," said Sabet. "By focusing on this signal-rich
point of light, we extended the LOD by two orders of magnitude."
Winny Tan, Ph.D., also a lead author and a lab member, said the
proof-of-principle tests of the sensor currently take between 30
minutes to an hour to complete. But she noted that this figure
is a bit misleading. "About 90 percent of our time was spent
in sample preparation, not actually performing the assay," she
said. "With further integration and automation of the test,
the time could be reduced significantly."
The laboratory already has developed a saliva-based electrochemical
sensor, which binds the protein of interest using an electrical
sensor system. Dr. Ho said the optical and electrochemical sensors,
like all technologies, have their pros and cons.
"The optical sensor requires a more expensive set up because
of the confocal microscope," said Ho. "So, in a small
dental or doctor’s office, the electrochemical sensor generally
would be easier and cheaper to use. But to really push down the
signal to noise ratio, the optical sensor has the advantage."
Ho said the optical sensor might be better suited for use in a
more specialized central laboratory. "But the technology
is advancing so rapidly, it’s difficult to predict how the optical
sensor might be used in the years to come," said Ho. "At
this point, it has certainly pushed the envelope for the limit
of detection and this will be an important capability in advancing
The National Institute of Dental and Craniofacial Research (NIDCR)
is the Nation’s leading funder of research on oral, dental, and
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Medical Research Agency — includes 27 Institutes and Centers
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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.