Forensic Sleuthwork in the Nano Realm

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By Julie West; cover photo by Julie West

Cocaine.  Methamphetamine.  Nitroglycerine.  Dichlorodiphenyltrichloroethane (DDT). Every explosive, drug, and biological agent carries a unique chemical signature. Forensic scientist and University of North Texas chemist Guido Verbeck studies these signatures to glean important information about a substance’s origin, composition, and distribution. Using a sophisticated instrument and method of his own design — the “nanomanipulator” — Dr. Verbeck can extract and analyze chemical residues from between the ridges of fingerprints down to the nano scale. The capability to study content at dimensions smaller than one-billionth of a meter offers clear advantages for forensic analysis. Interested parties, from Homeland Security and the U.S. military to the criminal and medical communities, are eager to use this device in the field.

The research grew out of a “Biometric and Forensics Summit” conference sponsored by the U.S. Army Research Laboratory that Verbeck attended in spring 2010. Participants were gathered to learn about fingerprint and facial recognition capabilities — how to analyze fingerprints and test for drugs and other illicit compounds.  “They put this incredible session together where the warfighters were in the same room with the researchers, and we had an open forum.  Researchers don’t typically communicate well with end users.  We’re concept people,” Verbeck confesses. 

But the meeting got both sides talking.  One of the soldiers wondered if explosive scraps found on the battlefield might have fingerprints, chemical residues and other biometric identifiers that could be extracted in small quantities and analyzed. Verbeck grins, “I knew this was the perfect job for the nanomanipulator.”

How it works

Nanomanipulator
photo by Julie West

Nanomanipulator.  The name conjures an image of a miniature, machine superhero from a comic book series.  Yet, the instrument is a superhero, of sorts. Petite, portable, and built to be mounted on any microscope and mass spectrometer, it is a sophisticated, in-hand tool for use in the field — able to detect compounds on nearly any material, including fabric, wood, plastic, skin, and metal.

Its central feature, a 100nm piezo-electric device with gold-plated probe*, is prefilled with a microextraction solvent and directly connected to a pressure injector. A joy-stick allows full range exploration across the X, Y, and Z-axes, while an attached microscope monitors every detail, producing high-resolution images of the nano terrain. The gold tip enhances the signal, allowing ultra-trace amounts of chemicals to be detected more effectively. Poised over its target, the probe swiftly extracts the particles in question.

The nanospray tip is landed within micrometers of the analyte particles ready for the injection/extraction —photo courtesy of Guido Verbeck

 

It’s all percentages

Nanospray ionization mass spectrometry is the next step in the research process. Used to analyze the extract, it produces a molecular “spectra” of ingredients — a precise blueprint of chemicals. Whether the substance is methamphetamine made in a bathtub in the U.S. or other illicit chemistries made abroad, Verbeck explains that it is possible to connect people and places through the chemistry. The process of reproducing illegal substances is difficult because there are impurities in the solvents, differences in the cook time, and other variables that affect consistency. But Verbeck’s research collaborators have designed algorithms that compare mass spectrometry measurements across databases, including those created by Advanced Chemistry Development Labs, a chemistry software company that supports R&D efforts. “The comparisons reveal the methamphetamine peak and any impurity peaks that are with it, and the relative intensity of those peaks — whether a substance gets cut down the line or changed — will remain. It’s percentages.”

The MS–MS spectrum of rock cocaine. The MH+ peak of m/z 303.80 appears along with the degradation products. The inset displays the expanded view of the degradation products occurring at a lower intensity.

A map of valuable information emerges that, from an intelligence point of view, Verbeck believes is more important than the ability to trace a substance back to the original manufacturer. If military investigators pick up chemistry of interest in one city and another in another city and the two chemical profiles match each other, then they know that the two persons are potentially connected. “We know who’s communicated or who’s touched the same explosive or passed it down the chain because the chemistry is connected. That’s the chemical signature.”

It is no easy task to retrieve analytics from fingerprint impressions. Most instruments cannot detect drug and explosive residues in ultra-trace amounts, and multiple factors hinder a reliable analysis of chemical composition. Nanomanipulation-microscopy coupled with mass spectrometry offers a superior solution.  The technology can lead investigators to potential suspects in terrorist attacks and other illegal activities.

On the go

Guido Verbeck, Assistant Professor of Chemistry, with the miniature, mass spectrometer
photo by Julie West

Using a grant from the U.S. Department of Defense, Verbeck is developing a small-scale forensic workstation for use in military field labs.  “The goal was to make a complement of instruments that are exploratory lab friendly for travel that you could put on a truck going to Afghanistan or any off-site location and know that it will work.” Verbeck says. 

Scale and weight matter. Instruments need to be much smaller than those in standard laboratories, so he experimented with the concept of a “pop out lab” — essentially taking a rack mountable system and reducing it to a small, one-drawer system.  The ability to micromanufacture is critical. “To make a mass spectrometer well, the machining, the tolerances and other components, all have to be very good or the electrical field will change. You need microfabrication techniques that are accurate and reproducible.  These are being developed by my team.”

Verbeck anticipates the first workstations will be ready for deployment to Afghanistan in February, 2013.   Once they are placed in field laboratories abroad, the portable tool kits must withstand extreme temperatures and constant dust — realities that can challenge even the finest crafted instruments. Time will tell how the portable tool kits will function in harsh conditions, but the trials provide an important measure of success.

Biological Applications

Like most superheros, the nanomanipulator has many powers. Verbeck explains that the device has been successfully applied to other disciplines, too. His collaborations with UNT biologists predate the defense and criminal applications. UNT plant biologist Kent Chapman approached Verbeck about the prospect of using an instrument that could manipulate single plant cells. The nanomanipulator was the tool for the job.  “The fact that we can go into fingerprint ridges means we can also go into single cells. The biochemical potential is huge,” Verbeck emphasizes. “This research could significantly enhance understanding of plant and animal physiology.”

Patrick Horn, UNT doctoral student; shown here with the nanomanipulator
photo by Jonathan Reynolds

Verbeck and Chapman, together with a team of master’s and PhD. students, developed a technique to extract single organelles out of cellular media and analyze them with mass spectrometry. This capability has shown promise in diverse biological research areas. Patrick Horn, a UNT doctoral student working under Chapman and Verbeck’s direction, has extended this research to study lipids — small molecules of fatty acids and derivatives, such as oils, waxes, sterols, and triglycerides, that are stored in the body for energy. Just as mass spec can provide a chemical profile of illicit substances, it can provide a map of cellular structures at the molecular level.

Isolated lipid droplets from Arabidopsis thaliana seeds stained with a fluorescent dye (BODIPY 493/503). Scale bar is 10 microns.
photo courtesy Patrick Horn, UNT Department of Biological Sciences

Since lipids are present in all organisms, Horn hopes that the ability to visualize the chemical composition of lipid droplets will shed light on disease and human health.  "Understanding how lipid droplets form and function has major significance," says Horn. "Lipid storage and mobilization underlies human health issues, such as obesity and diabetes. The ability to study these structures on the nanoscale may result in ways to modify how fats are stored and burned."

The team’s research has been published in the Journal of Biological Chemistry and was recognized by the Faculty of 1000, an organization that identifies and evaluates the most important articles in biology and medical research publications. Horn began his research on lipids in fall 2008, and he says that he'll continue working with Chapman and Verbeck until his scheduled graduation in 2013.

Verbeck is partnering with well-known lipid scientist Al Merrill at Georgia Tech to apply these biochemical techniques to mammalian cells. Their experiments involve a class of lipids called sphingolipids, which are believed to play an important role in signal transmission and cell recognition. The major focus of this research involves the lipid backbones of sphingolipids that regulate cell behaviors.

When mounted on the gold tip of a nanomanipulator and suspended over a cellular medium, specific sphingolipids cause cells to stop their normal meiotic behavior, i.e., the cells will not divide until they destroy themselves. Naturally occurring biological mechanisms, such as E. coli, produce this reaction.  By simulating this process in a laboratory, monitoring the chemistry that crosses the membrane, and then extracting the chemistry from the cells, Verbeck and Merrill can learn about the relationship of mitosis to disease.

Mercury toxicity in livers of northern pike from Isle Royale, Michigan. Lightly-colored, pink livers (A) had low total mercury concentrations and darklycolored,
red to brown livers (B) had high total mercury concentrations.
photo courtesy of Aaron Roberts

The alliance of nanomanipulation with microscopy and mass spectrometry proves useful in the study of environmental contaminants, too. Aaron Roberts, assistant professor of biological sciences at UNT, studies mercury concentrations in fish and tags lesions in their livers using these tools and with the help of Verbeck’s forensic expertise. “If there’s chemistry there, we can extract it and look at it, down to an obscenely small scale,” says Verbeck.

Laser ablation — a process that irradiates a solid with a laser beam — allows Roberts to extract chemistry directly from the liver tissue into the mass spectrometer. Roberts examines the healthy fish livers as well as the livers with lesions. Correlations are then run using data collected from the areas with lesions against those where the mercury was located. To the team’s surprise, initial experiments revealed that the highest concentrations of mercury were localized in the lesions.  Subsequent tests confirm these findings. “This is the first time anyone has ever seen this,” says Verbeck. The research has been well received in the science community. Roberts states, “The research has the potential to impact our understanding of the transport of mercury in foodwebs, the toxicology of methylmercury, and to better inform our ability to assess mercury ecological and human health risk.”

Saving lives

Deployable, or portable mass spectrometer with views of pump and inlet
graphic courtesy of Guido Verbeck

Verbeck has developed other miniature, portable forensic instruments that offer equally potent solutions for real world problems.  One such device, the portable mass spectrometer, is a softball sized ion detector that samples the air for a variety of chemicals and transmits the data to field stations. The deployable balls can go where humans cannot or should not go, such as hazardous environments.

Originally developed by Verbeck for the International Space Station, the tool is useful for analyzing conditions in space but can also alert the military, police, and other government agents to situations involving bio-weapons or harmful toxins. If poisonous chemicals are present in the air or soil, this capability can avert danger and save lives.

1st Detect Corporation, an Austin-based satellite and space research company, has partnered with Verbeck to commercialize the technology so that diverse groups can successfully deploy the device. Verbeck’s expertise in mass spectrometry and product miniaturization is key for success.  "This technology allows us to put what was once a cumbersome piece of scientific equipment into a portable device that is useful in many applications," says Verbeck. "Otherwise, field use of mass spectrometry is just not practical."

Another innovative use of the miniature spectrometer concerns fire hazards. Verbeck got the idea when he met with Shreveport firefighters in a forum akin to the one offered by the U.S. Army Research Lab conference, but this time his father, a chaplain with the Shreveport fire department, provided the impetus.  “Dad said I should see the gear the firemen have to wear in order to go into a fire. They have no way of knowing what’s burning in there, and they have to prepare for the worst.  They typically carry 80 pounds on their back, which makes it difficult to maneuver.  If the firefighters had a way of knowing ahead of time what was burning inside, they could adjust their strategies,” Verbeck says. Industrial fires, for example, are managed very differently from a typical house fire.

Inspired to help, Verbeck devised a cheap, portable, ion trap mass spectrometer that can be thrown or dropped into a burning building and quickly deliver the chemical makeup of the surrounding atmosphere.  Fire chiefs can decide a course of action depending on what’s burning.  Even first responders on the scene can use it for a quick handle on the nature of the fire.  The instruments surprisingly endure the extreme heat long enough to gather and transmit the data before they eventually succumb to the fire.

A solid foundation

What’s next? Verbeck and colleagues from the departments of Biological Sciences, Chemistry, and Electrical Engineering are expanding the forensic initiative at UNT.  They’ve formed a research group, Forensic and Investigative Science and Technology Instrument Development, with the aim to promote greater collaborations among the broader science and engineering communities, new technology research, and facility expansion. UNT has designated this group’s work to be an important “strategic area of investment” in alignment with the research cluster expansion.

The existing forensic science program, one of the few certified programs in the U.S., offers the largest mass spectrometry laboratory for research and service in the North Texas and Oklahoma area. Equipped with an administrative commitment of infrastructure and other resources, the team will greatly augment the program with multidisciplinary approaches in the forensic sciences toward research and the development of detection sensors and instrumentation. 

Meanwhile, Verbeck looks forward to working in his new laboratory in Hickory Hall at UNT.  The new location brings a clear advantage over the lab’s current home: the solid, bedrock foundation is essential to reduce the risk of vibrations and instrument drift. Verbeck has built a solid foundation of research at UNT. His projects are garnering considerable attention from chemistry peers, industrial and federal agencies, and his colleagues and students see him as a gifted teacher as well as a talented chemist. The UNT Office of Research and Economic Development recently awarded him with a “UNT Early Career Award for Research and Creativity” for his accomplishments. His research initiative, creativity and intellect demonstrate that he is on his way to being one of the leading researchers in the design and development of new analytical instrumentation and methodologies.

In closing, Verbeck reflects, “No matter how transformative new developments in Forensic Science may be, research is never terminal.  As small platform, forensic research improves, the question becomes a matter of ‘how little’ or ‘how much’ information is needed to convict?  Issues such as this affect the greater community, and scientists such as myself are aware of these challenges as we continue to transform our laboratories and develop smaller instruments.”

* developed by Diagnostics and Characterization Group for use on scanning electron microscope manipulation

— submitted January 2012

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