Alan Dove, PhD
Researchers are creating sophisticated pathogen-detection systems for bioterror defense. But will this lab technology work in the field?
In April, 1979, workers at a secret Soviet biological weapons facility neglected to replace a filter in a laboratory ventilation system, and a cloud of highly weaponized Bacillus anthracis quickly spewed into the air outside. The deadly plume drifted downwind, infecting humans and cattle across a wide area. Russian news sources
attributed the resulting outbreak—which ultimately killed more than 64 people—to tainted meat, but American officials were skeptical. It wasn’t until 1992 that Western scientists were finally allowed to visit the site, determine the true cause of the outbreak, and glimpse the vast scope of the former Soviet bioweapons enterprise.
The collapse of the Soviet Union reduced that particular threat, but as governments, companies, and researchers wrestle with new fears of bioterrorism and emerging diseases, the problem of identifying and tracking pathogens remains acute. Using new technologies and strategies, several corporate and basic research efforts have made promising progress in the field recently, while also uncovering new challenges.
Sample me softly
For biowarfare agents and some naturally-emerging diseases, one of the first diagnostic challenges is simply collecting the sample properly. To find aerosolized agents such as weaponized anthrax, current systems often rely on pulling air through a fine filter—a strategy atmospheric physicists have used to isolate pollutants for decades. Unfortunately, that may not be the best strategy for collecting live microorganisms.
“Within aerosol research, we’re . . . not concerned about whether or not a particle is alive or dead, so that is kind of unique to bioaerosols and biodefense, and it’s a really important issue,” says Timothy Buckley, PhD, associate professor of environmental health sciences at The Ohio State University, Columbus, Ohio. Indeed, when Buckley and his colleagues recently tested different sampling filters, they found that two of the most commonly used systems reduced the test sample’s culturability three- to six-fold.
To address this problem, both Buckley and Ana Rule (a colleague who is now a postdoctoral researcher at the Johns Hopkins University Bloomberg School of Public Health, Baltimore, Md.), are studying a different kind of filter called the BioSampler. Rather than using a fine mesh, the BioSampler uses a gel-like liquid to capture aerosol particles. In side-by-side comparisons, the BioSampler kept nearly all of the bacterial cells in a test sample intact and culturable.
While the BioSampler is fast becoming the standard in environmental health research, Buckley concedes that it has some disadvantages that could keep it out of field-deployed pathogen sensors. “Anytime you have a fluid-filled sampler, you have to be concerned about its orientation and the loss of that fluid, so I think it does pose some logistical issues,” says Buckley.
For researchers, though, the system’s other advantages offset its more complicated maintenance requirements. “Even though it’s a bit more cumbersome to manage in the field, once you have the sample you can now remove aliquots from the sampler and plate them, [or] use them for flow cytometry,” says Buckley.
click to enlarge
This schematic illustrates the principle of Nanosphere’s Verigene system for bioterror pathogen detection. The detection system is based antibody-coated, barcode-labeled Gold nanoparticles. (Source: Nanosphere)
While a sampling system certainly needs to be efficient, another key feature for field equipment will be user-friendliness; it needs to be usable by postal workers, military personnel, or first responders without specialized scientific training. To that end, computer scientists at Los Alamos National Laboratory, Los Alamos, N.M., have been refining a device they call the Hands-Off Sampler Gun.
With a sampling pad, digital camera, voice recorder, and a global position system (GPS) receiver all built-in, the Sampler Gun documents the entire collection process for each sample. Besides preserving key information that field workers might forget to record, the device also provides a solid chain of custody in case the evidence is needed in a criminal trial. The Federal Bureau of Investigation (FBI) plans to begin field tests on the device early next year.
While getting a good sample into the device is clearly important, the business end of a biological agent detection system is always its assay. In recent years, researchers from fields as diverse as nano-engineering and immunology have focused their attention on developing new bioweapons detection assays, producing a flood of promising designs. Unfortunately, nearly all of these methods still suffer from serious drawbacks.
“In my opinion, there is not a single winning system at this point. Detecting systems currently being employed are rapid and easy to use, but nonetheless they do have a high rate of false-positive[s],” says Jeffrey Tok, PhD, staff scientist in the chemical biology and nuclear science division of the Lawrence Livermore National Laboratory, Livermore, Calif.
Tok and his colleagues recently completed a review of several of the leading biodefense assay systems, and found that while all had potential, none were ideal. “The ultimate test will be rendering all the available technologies reported in literature to be field-deployable, where the conditions are significantly different from a laboratory setting,” says Tok.
Besides hardening the systems against environmental fluctuations, companies hoping to build biodefense detectors also have to make their products easy to maintain and their readouts easy to interpret. In addition, Tok points out that many of the published strategies rely on antibodies as sensing ligands. While antibodies can be exquisitely sensitive and selective, they are also costly and relatively fragile.
Nonetheless, even Tok and his colleagues often rely on antibodies in their own assay systems, preferring to focus on the problem of user-friendly readouts. In their most recent work, the Los Alamos researchers have developed a technique for attaching nanowire “barcodes” to antibodies or other biomolecules, allowing them to be tracked optically or electrically.
The nanowires consist of segments of different metals, which can be placed in unique sequences to encode labels. Using a segment of gold to indicate a binary “0,” and a segment of silver to indicate a “1,” the investigators can construct nanowires with antibodies attached only on the “1” segments. Free-floating fluorescent secondary antibodies in the same solution highlight all of the wire segments that have bound their target antigens, providing a simple optical barcode. In the readout, a completely dark wire has not found its target in the test sample, while a wire with a single light band near one end might indicate that the antibody coded 010000 has found its target. Because it can identify multiple antigens simultaneously without specialized hardware and software, Tok hopes the system will be more practical than some array- and bead-based assays.
Hey Sarge, what’s this powder?
As researchers continue to develop new assays, companies working on biodetection systems prefer to apply platforms they were already developing for other markets, such as clinical diagnostics. Nanosphere, Northbrook, Ill., typifies this approach. “We have a platform for diagnostics that’s based on nanotechnology, using gold nano-particles for targeting infectious diseases,” says William Cork, Nanosphere’s chief technology officer. Cork adds that the company has also adapted the system, called Verigene, to diagnose genetic disorders and identify protein biomarkers, primarily for hospital diagnostic laboratories.
In the Nanosphere system, gold nano-particles conjugated with either antibodies or oligonucleotides target the antigen or sequence of interest and an integrated microfluidic device analyzes the
nano-particles to detect binding events. With current miniaturization technology, the company can put the hardware and reagents for testing as many as 46 different analytes into a single disposable cartridge. Plugging different cartridges into the basic Verigene unit provides a lab with a wide range of testing options.
While the principal market for these assays is still clinical labs, Nanosphere submitted a proposal for developing field-ruggedized biodefense assays after the 2001 anthrax attacks in the US. Of approximately 12,000 applicants for the multi-million dollar contract, Nanosphere won.
In addition to its standard clinical Verigene system, the company now manufactures a more durable version for use by military and law enforcement personnel. In a typical scenario, a specialized military biodefense team might be called to a field position to investigate a suspicious substance. After donning protective equipment and collecting a sample, the team can insert it into the Verigene system and get an analysis.
“We continue to have contracts with the Department of Defense, who continue to add applications to the platform,” Cork says. In the most recent contract, the company will receive $1.2 million to add two toxin assays to the military Verigene assay.
The grants are certainly useful for a biotechnology company, but so far, the final market for biowarfare detection systems remains minuscule. “Across the biowarfare industry, in terms of detection platforms, there isn’t a significant amount of sales over the last 4 years,” says Cork, adding that “quite frankly the use of them in the field is quite limited.” He estimates that total sales of bioweapons detection systems have been in the “tens to hundreds” of units, or a tiny fraction of the clinical diagnostic market.
Despite the slow uptake, researchers and companies working in the field continue to refine their pathogen detectors. The devices may never be—or need to be—as ubiquitous as smoke detectors, but if the remaining bugs can be worked out, they might eventually become common sights in public places. After all, it’s better for an airborne biowarfare agent or emerging virus to drift into a sampling cartridge than a nose.
About the Author
Originally trained as a microbiologist, Alan Dove has been writing about science and its interfaces with industry and government for more than a decade.
This article was published in Drug Discovery & Development magazine: Vol. 10, No. 9, September, 2007, pp. 22-24.
The May, 2007 issue of Drug Discovery & Development featured a related article “Big Biodefense Goes Small,” which profiled some of the top players developing countermeasures against bioterrorism.
Filed Under: Drug Discovery