The vast scientific and medical knowledge accumulated in the last quarter century has opened new doors for disease diagnosis and control. Researchers now have the ability to work in the nano realm, a world so small that individual molecules can be manipulated not only to detect disease and other foreign agents immediately, but also to develop unprecedented measures for treatment and prevention. The ability to recognize the specific molecular structures of pathogens, viruses and DNA sequences is critical in this process.

Richard Willson, professor of chemical engineering and professor of biochemical and biophysical sciences at the University of Houston, has spent nearly two decades working to identify and analyze individual protein, DNA molecules and disease organisms, a research area known as molecular recognition. For instance, Willson and his team are able to determine if a particular antibody or DNA molecule will consistently bond with a disease-causing virus or toxin, and to characterize the speed and energetics of the association. If the research can prove that this bond is formed on a regular basis, then scientists can harness the molecular recognition for diagnostic or treatment purposes.

In an application funded by the Homeland Security Advanced Research Projects Agency, Willson is working with Yuriy Fofanov, assistant professor of computer science at UH, (who Willson says has developed “the world’s best algorithms for exhaustive searching of genomes”) and George Fox, professor of biology and biochemistry and professor of chemical engineering, (who co-discovered the Archae, one of the three classes of living things on the Earth) to identify and validate specific DNA probes for organisms of biodefense interest. In other words, if the research identifies a specific DNA probe sequence that will bond to the anthrax virus, and officials fear anthrax is present in an area, researchers could use that DNA probe to verify or disprove the presence of the disease, enabling authorities to take appropriate measures quickly, if necessary.

“Biological molecules can be amazingly good detectors, capable of recognizing one type of bacterium or virus in the presence of its closely-related ‘cousins’, or a particular genetic sequence out of millions,” said Willson. “The air in Houston and every major U.S. city is routinely monitored for biothreat agents and a false alarm is just unacceptable in a civilian application.”

Willson and his team are working to understand the structural and energetic basis of specific interactions between molecules in order to make these interactions useful in medical diagnostics, purification and biodefense. Specifically, Willson’s research revolves around characterizing and improving the ways detector molecules, such as antibodies and DNA probes, recognize their targets, and using these interactions to purify biomolecules. He will also be working to improve ways in which a detector will recognize its target.

In one of many ongoing research projects, Willson and his team are working with Paul Ruchhoeft (1998 MSEE, 2000 PhD EE), assistant professor of electrical and computer engineering, to develop a new biomolecule labeling system and diagnostic tool utilizing optical signals returned from corner-cube retroreflectors.

“Most people are familiar with the related safety technology, which makes a jogger’s shoe heels shine in your car’s headlights at night,” says Willson, “but with modern fabrication techniques developed by Ruchhoeft and John Wolfe, professor of electrical and computer engineering, we can use retroreflectors for a whole range of new biological and biomedical applications.”

Preliminary results in manufacturing micro-retroreflector cubes, showing a Birdseye view (a) of the lithographic pattern in resist, as printed with AAL, and (b) a view of cubes that have been “scraped” off of the surface using a scalpel.

Specifically, these retroreflectors are tiny cubes, mirrored on three sides, that return light back in exactly the same direction from which it originated, making them extremely detectable even through tissue or at a distance. Retro-reflectors have long been used in both scientific and consumer applications. In fact, the Apollo program placed retroreflectors on the surface of the moon as part of ongoing experiments on lunar orbital dynamics. Commercially, retroreflectors are utilized daily as road lane markers and on running shoes, safety vests, reflective road signs and bike pedals, but have not previously been used in bioanalytical methods.

The team will construct self-assembling, chemically responsive micro-retroreflectors used for remote monitoring of toxic gases or bioagents for biodefense. With the ever-present danger involving the detection of such agents, newer technology is needed to safely and carefully locate areas affected by toxins.

Because of their extremely high detectability, retroreflectors can be utilized as a low-cost diagnostic tool with many possible applications, such as the detection of antibody-targeted colon cancer markers and continuous glucose level monitoring in diabetic patients. Currently, typical diagnostic sensors and the equipment that read the output signals are relatively bulky and expensive, making continuous monitoring of biomarkers impractical outside a laboratory setting. Working in the nano realm allows researchers to produce a more readable, more accurate signal from an extremely small sensor, allowing doctors and patients to gain a more complete understanding of the disease or condition being treated.

So, how does it work? Willson explains that one side of the micro-retroreflector will be coated with antibodies but missing a mirror, deactivating the reflector until the target molecule (e.g., a bioagent) “bridges” gold nanoparticles with the surface, assembling the third mirror and restoring retroreflectance. If a bioagent exists in an area, scientists will observe a reflection in response to the binding of the antibodies and target molecules.

“If we can monitor the dangerous elements continuously, at a distance, then we have accomplished a great deal,” says Willson.

Such self-assembling micro-retroreflector technology could also be used in a new class of implanted, long-term, continuous glucose monitors in which patients and doctors could detect the retroreflectance through the skin using inexpensive LED optics. Continuous glucose monitoring is a long-sought goal in diabetes
treatment. Recent clinical recommendations emphasize the value of testing glucose levels far more frequently than most patients do now.

“For the treatment of diabetes, the development of better monitoring technology is crucial, because right now doctors and patients don’t have a practical and reliable way to monitor glucose levels continuously,” Willson explains. “Without this ability, doctors cannot treat the disease as effectively as they could with the additional information our self-assembling micro-retroreflector technology may allow.”

The micro-retroreflectors designed by the team for biomedical purposes will be only a few micrometers wide (smaller than a red blood cell), which will allow the biological sample doctors need for analysis to be very small. For long-term monitoring uses, the micro-retroreflectors could be designed as a non-irritating implant that displays the needed signals through the patients’ skin or in a pill form for ongoing diagnoses in real time.

In addition to retroreflectors, Willson is working with atomic lithography pioneer Wolfe and Dmitri Litvinov, associate professor of electrical and computer engineering, to develop a nanomagnetic sensor array capable of detecting single molecules using the sensor technology at the heart of high-density magnetic disk drives like that in the iPod. While the biomolecular recognition technologies today can carry out
genome-wide profiling of clinical specimens, they require relatively large samples, which are often not readily available, especially in medical applications. Researchers currently have a critical need for new technologies that enable clinical specimen analysis of ultra-small samples.

“These joint research endeavors enable us to utilize the expertise of scientists from a variety of disciplines to achieve more than any of us could alone,” says Willson.

Recently funded by the National Institutes of Health (NIH) for $891,000, the group will develop an ultra-sensitive, highly-stable nanomagnetic sensor array that is capable of evaluating a variety of samples, outputting real-time, high-quality data.

Such a nanomagnetic sensor array will capitalize on dramatic advancements in magnetic disk data storage technology, as represented by Litvinov, who has successfully implemented a number of nanomagnetic concepts in commercial magnetic data storage systems. These concepts can be integrated into a practical sensor array with extremely high densities of individually addressable sensors.

Specifically, the sensor array will be comprised of a million giant magnetoresistive (GMR) sensors, all housed in a single square millimeter of space. In a disk drive, one GMR sensor will scan the surface of a disk bearing tiny magnetic domains. In the biomedical sensor, the many GMR sensors detect magnetic nanoparticles that act as labels allowing detection of individually-labeled molecules. Between the nanoparticle and each GMR sensor, Willson’s group will incorporate biorecognition molecules with specific binding capabilities.

Professor Richard Willson, PhD student Maria Añez (2004 MSChE) and postdoctoral researcher Ekaterini Kourentzi (1999 MSChE, 2002 PhD ChE) determine the composition and purity of RNA and protein samples.

In a specific application of NIH interest, the sensor can be used for massive screening of drug candidates for their ability to block interactions involved in disease processes. For example, the sensor array can be decorated with a cell surface protein known to be an entry receptor for a virus such as influenza or HIV. A magnetic nanoparticle bearing one or more copies of the virus’ cell-binding protein will bind to the cell protein and to the GMR sensor, producing a high-quality signal that informs researchers whether the molecules have associated. The sensor array allows the simultaneous testing of an enormous number of drug candidates for their ability to block virus/cell association.

In a separate application funded by the Alliance for NanoHealth, the magnetic nanosensor can be adapted to perform molecular diagnostic assays on clinical biopsy specimens, especially in the molecular diagnosis of cancer. A particular goal is to obtain more useful information from the standard “fine needle aspiration” procedure, which uses a fine gauge needle to sample fluid from a breast cyst or remove clusters of cells from a solid mass. This procedure is widely used in part because it is less invasive than many other methods, but the quantity of sample obtained is too small to be used in many of the most useful molecular diagnostic assays.

The nanosensor will be used to test breast cancer markers including the estrogen receptor (ER). The estrogen receptor is the most important growth factor identified for breast cancer; 50 percent of primary breast cancers in women are ER-positive, while most normal breast tissue and benign breast lesions lack the receptor protein. Hormonal therapies for breast cancer have had a bigger impact on recurrence and survival than any other treatment.

“Breast cancer detection is one of many biomedical applications this sensor array can be used for,” said Willson. “Once developed, we will be able to detect a variety of disease biomarkers and pathogens in the body, screen for likely drug candidates and look for the presence of bioterrorism agents.”

Willson’s team is especially well-prepared to capitalize on the synergy of recent advances in nanotechnology, the explosive growth in genomics and proteomics and the unique nanofabrication capabilities at UH. In addition, the college’s strategic location near the world-renowned Texas Medical Center allows further collaboration among the biosciences, engineering and medical disciplines, specifically between researchers at UH and M.D. Anderson Cancer Center, Baylor College of Medicine and the Methodist Hospital.

In addition to the National Institutes of Health and the Alliance for NanoHealth, Willson’s research is funded by the Welch Foundation and NASA.

More articles:
• Biodetection: Making Life Better One Molecule at a Time
• Crystal Clear Diagnosis: UH Researchers Use Lasers and Nanocrystals to Detect Diseases
• Tiny Tools Cover Lots of Ground: UH-Developed Nanodevices Search for Thousands of Diseases