By Neal Singer
Who would have guessed that when the Star Trek medical diagnostic tool known as the tricorder makes its appearance in real life, the first user might be . . . your dentist.
According to a paper in the March 27 PNAS (the Proceedings of the National Academy of Sciences), a recently completed pilot study conducted with the University of Michigan shows that a Sandia-developed handheld device determined in minutes — from a tiny sample of saliva alone — not only if a patient has gum disease but quantitatively how advanced the disease is.
“The gold standard for any medical test is when instruments are used to examine human patients,” says Sandia researcher Amy Herr (8321). “The pilot study allowed us to compare our results to accepted clinical measurements. Then we could statistically validate both the perio-dontal disease biomarker and the new microfluidic instrument.
“We achieved faster and more reproducible results because we combined steps that ordinarily require time-consuming manual handling by many people, into a single automated device.”
Because the amount of sample fluid needed for testing is so small, Amy sees further applications in other disease areas — including potentially improved diagnosis of prostate and breast cancer — as well as rapid measurements of serum in animal models employed in vaccine development research.
Says Sandia researcher Anup Singh (8321), “This technology also has great promise for
Sandia’s efforts in homeland defense. We have on-going efforts to use the diagnostic platform to detect biotoxins and other markers in bodily fluids to be able to diagnose exposure to a biological agent.”
“We’ve filed patents and technical advances to protect the work,” Amy says. “The study has sparked commercial and university interest in our inventions. Our team — an interdisciplinary group of internal and external collaborators — believes Sandia’s contributions in this area could advance personalized medicine. So we’re motivated to extend the limits of Sandia’s lab-on-a-chip tools.”
A “lab on a chip” refers to an entire automated laboratory on an area the size of a computer chip, able to perform chemical analysis on minute amounts of material.
How it works
While components of the saliva-detection technique were reported earlier by Sandia, this is the first comprehensive study of Sandia’s integrated clinical method.
The basic principle? “Biomedical researchers have suspected that changes in the amount or type of proteins present may be useful as biological markers in disease diagnosis,” says Amy. “Our current work with a particular enzyme in saliva supports that hypothesis regarding perio-dontal disease.”
Aiding dental practitioners, the pocket-sized device measures the state of biomarkers to determine how much the disease has been set back. Its progress may be cloaked, silently advancing or retreating without showing any signs.
“Periodontitis can be episodic in nature,” says Amy. “You need to know the stage of disease progression to diagnose and treat the illness most effectively. The enzyme [biomarker] that we monitored decreased or stabilized if the treatment was working well.”
Often, owing to the time and expense involved, practitioners formerly had not been able to perform extensive biochemical investigations.
The work, funded by the National Institute of Dental and Craniofacial Research (NIDCR) — one of 20 institutes in the National Institutes of Health — is the first application using microliters of saliva, a painlessly and easily secured fluid. The real-life alternative for the most part has been quasi-subjective physiological measurements, such as gum recession and gum bleeding on probing, to diagnose periodontitis.
Unlike Sandia’s MicroChemLab — its patented version of a lab on a chip — which reports multiple protein signatures in fluids of interest, the clinical diagnostic instrument described in PNAS is a lab on a chip designed to quantify the amount of a specific protein (or panel of proteins) present in particular biological fluids. Monitoring quantities of specific proteins makes the tool useful as a clinical diagnostic.
Using a disposable lab-on-a-chip cartridge, the device makes use of a molecular sieve made out of a polyacrylamide gel. The location of the sieve in the microfluidic chips is determined using photolithographical methods adapted from the semiconductor industry. The gel is porous, with very small openings. A low electrical current (measured in micro-amps) is passed through the gel and a process called electrophoresis moves charged proteins through it. The gel has a Jell-O-like consistency and, by permitting the easy passage of smaller molecules and slowing the passage of larger ones, quickly separates proteins contained in the saliva. Prior to this separation, the proteins are brought into contact with specific antibodies chosen for their ability to bind to the biomarkers. The antibodies are prelabeled with fluorescent molecules attached to them. Interrogation by laser of these combined molecules — fluorescent antibody and fluorescent antibody bound to the biomarker — determines the amount of biomarker present, indicating the degree of periodontitis.
Sandia authors of the study, in addition to Amy and Anup (the NIDCR project primary investigator), include Anson Hatch, Daniel Throckmorton, James Brennan (all 8321), and Huu Tran (8755), as well as Will Giannobile of the School of Dentistry at the University of Michigan, Ann Arbor.
A new type of electrochemical sensor that uses a unique surface chemistry to reliably and accurately detect thousands of differing biomolecules on a single platform is being developed by a Sandia research team led by Susan Brozik (1714).
The new bioagent detection system could be applicable in homeland defense, safeguarding warfighters, and clinical diagnostics.
“A problem with the majority of existing biosensors is that they only look for one type of biomolecule [DNA or protein] at a time,” says Jason Harper (1714), research team member. “This can often lead to inaccurate or inconclusive results and limits the use of the sensor. Where our sensor differs is that multiple characteristics of several bioagent targets can be tested on a single chip. Identification of several DNA sequences and protein markers are needed for detection of multiple targets and will allow for accurate discrimination between similar bioagent threats.”
For example, instead of using only an antibody that binds to the surface of an anthrax spore, the new Sandia sensor could test for several DNA sequences and internal and external proteins unique to anthrax. This provides numerous positive readings for the target agent or agents, significantly increasing confidence in the sensor results.
Will simultaneously detect thousands of biomolecules
The new Sandia sensor will be able to simultaneously detect thousands of biomolecules on a single platform. By integrating antibodies, DNA, and other biomolecules on a single device, the number of lab instruments, volume of reagents required, time for analysis, and the cost of effectively performing thousands of tests are all reduced.
The platform, a microfabricated chip, is just one inch by one inch in size. Several technological advances in microfabrication processes have increased the numbers of electrodes that can be produced on a sensor platform. A major challenge is how to pattern different biomolecules onto closely spaced micrometer-sized electrodes. Brozik’s group believes the answer lies in the electrodeposition of aryl diazonium salts.
The surface chemistry, produced by team members David Wheeler and Shawn Dirk (both 1714), possesses several advantages over currently used chemistry, David says.
“This diazonium-based surface chemistry can be selectively deposited onto several types of substrates by controlling the charge of the substrate in the diazonium solution,” David says. “Because the deposition of the diazonium molecules is based on the application of an electrical potential, the selective patterning of individually addressable electrodes is possible. Upon deposition, covalent bonds are formed with the substrate, producing a highly stable film.”
Compatible with a wide variety of biomolecules
The chemistry is also compatible with a wide variety of biomolecules. DNA, antibodies, enzymes, and peptides all have been patterned onto arrays at Sandia using this chemistry.
After treating the sensor with the target solution, the array is washed and treated with a different solution containing molecules that bind to the other end of the target biomolecule, forming a “sandwich.” These secondary labels form an electroactive product that is detected by the electrode.
Says team member Ronen Polsky (1714), “We are also investigating a new electrochemical detection method, using electrocatalytic nanoparticles, that we hope will eliminate the extra washing and labeling steps. This will greatly simplify the end device.”
Some of this work was recently featured in an article in Langmuir, published by the American Chemical Society. Diazonium chemistry was used to selectively deposit the enzyme horseradish peroxidase, which was then used to electrochemically detect hydrogen peroxide.
Electrochemical detection holds many advantages over other common optical-based biosensors, Jason says. By eliminating optics and using semiconductor microarrays, the end device is smaller, more rugged, and simpler in design.
Eventually the sensor array will be integrated in a deployable electrochemical sensor that will have an electronic readout identifying the biomolecules detected, or wirelessly transmit the results to a computer or network. Reaching that point will take anywhere from two to five years, says Ronen.
Currently the sensor arrays in the project allow for selective identification of nine biomolecules, Jason says. However, the work has kindled the interest of commercial sensor companies. The Sandia team recently traveled to Seattle to test their surface chemistry on a commercial array produced by CombiMatrix, a company that specializes in producing semiconductor arrays with more than 12,000 individually addressable electrodes in an area less than one-inch square.
“The team successfully patterned peptide ligands onto 2,151 individual electrodes out of an array of 12,544 electrodes,” says Susan. “The resulting electrochemical signal from the captured peptide was used to pattern the Sandia thunderbird symbol as well as the CombiMatrix logo.”
Because of this initial success, Sandia and CombiMatrix are pursuing a cooperative research and development agreement (CRADA) for further development of a sensor using Sandia’s surface chemistry and Combi-Matrix’s electrode array, to ultimately test for thousands of biomolecules simultaneously. Funding for this research has been provided by Sandia’s internal Laboratory Directed Research and Development (LDRD) program, the National Consortium for Measurement and Signatures Intelligence (MASINT) Research, a Defense Intelligence Agency program that seeks to promote collaborated research among academia, industry, laboratories, and DOE. CombiMatrix is funded by the Defense Threat Reduction Agency.
Electrochemical sensor team members
A memorandum of understanding between Sandia and the University of Illinois at Urbana-Champaign was signed at an official ceremony April 3 at the Urbana-Champaign campus.
The agreement formalizes the relationship between the two institutions and describes common fields of research interest in nanoscience, cognitive neuroscience, information technologies, water technologies, high-performance computing, energetics/combustion, complex systems/system-of-systems, and high-frequency imaging and communications.
Sandia Senior Manager Russ Skocypec (6340), who earned his BS, MS, and PhD in mechanical engineering at Illinois, serves as Sandia’s lead representative involved with developing the agreement.
Dave Carlson, director of Nuclear Weapons Planning, Operations, and Integration Center 200, serves as campus executive for the U of I relationship. He says the agreement will allow Sandia and the university to develop and pursue joint research initiatives that leverage both institutions’ strengths and infrastructure.
“The general areas of collaboration include activities to support complementary institutional goals and share and leverage specialized research facilities and equipment,” says Dave, an Illinois alumnus with MS degrees in astronomy and nuclear engineering. “The agreement will also increase inter-institutional collaborative engagement of faculty, staff, and students.”
“By joining our resources together with those of Sandia National Laboratories, we can have a significant impact on an incredibly broad range of research,” says Ilesanmi Adesida, dean of the College of Engineering at the University of Illinois at Urbana-Champaign.
Pierre Wiltzius, director of the Beckman Institute for Advanced Science and Technology, notes that historically the University of Illinois and Sandia have had a very strong relationship in the physical sciences and engineering.
“We are very much looking forward to expanding our interactions with Sandia into new areas including cognitive sciences, neurosciences, and human and computer speech and vision,” says Wiltzius. “This expansion will also engage faculty and students from the College of Liberal Arts and Sciences and is squarely aligned with the strategic initiatives of the Beckman Institute and the University of Illinois.”
Sandia currently has 19 active agreements with 15 universities across the US. -- Stephanie Holinka