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Feature Articles : Nov 1, 2009 (Vol. 29, No. 19)

New Capabilities Fortify Biodefense Tools

Rise in Funding for Rapid Diagnostics Has Resulted in Innovative Biodetection Systems
  • Gail Dutton

Clinical detection methods are taking to the field, boosting the capabilities of point-of-care biodetection devices for clinical use as well as battlefield diagnoses. Speakers at Select Biosciences’ “Advances in Biodetection Technology” held in London last month outlined their work in developing fluorescent imaging for explosive particulates and several approaches to integrated PCR amplification and detection for point-of-care applications and environmental field work. Rapidity was a hallmark of most of the advances.

Chemists at the University of California, San Diego have developed a fluorescent imaging method to detect explosive particulates, like those left behind in the bomb-making process. Recently commercialized by RedXDefense, the system detects nitro compounds and either quenches or lights up the fluorescent sensor, according to William Trogler, Ph.D., professor of chemistry and biochemistry. The same technology also can be applied to hydrogen peroxide, an ingredient in many homemade explosives. Sensitivity is in the range of nanograms to picograms. “The advantage,” Dr. Trogler said, “is that it is a really inexpensive, intuitive visual indicator.” Results can be seen by black light, he added.

To advance that work, Dr. Trogler and his colleagues are exploring selectivity issues, trying to develop a sensor that also can identify specific explosives.

Another project, still in the early research stage, involves using hollow nanoshells as biodetectors.

“We’re looking at nanoshells as nanosensors in an aqueous environment,” he said. Specifically, a functionalized nanoshell with a hydrophobic coating could be used to separate and concentrate targeted analytes from solution, so the encapsulated polymer-based sensor could detect its presence as the nanoshell dissolved.

These nanoshells may also be used in vivo as probes and for target delivery. “We’re looking at them to deliver chemotherapy,” Dr. Trogler said. Because they are stable, they also may have promise as imaging agents.

At Network Biosystems, researchers are developing a fully integrated, “samples-in/results-out” microfluidic system that will allow multilocus sequencing of clinical or biothreat pathogens in complex samples.

“The environmental system would function autonomously in the field, and the clinical system would be automated and used by nontechnical operators in the emergency room or on the battlefield,” Richard Selden, M.D., Ph.D., executive chairman, elaborated. Therefore, ease of use is vital.

Real-time DNA sequencing has the potential to dramatically improve both clinical diagnostics and biodefense, he said, “by distinguishing between pathogens and their nonpathogenic near neighbors to generate actionable information.” Usefulness is further enhanced by speed. Samples-in/results-out time is about one hour, versus days to weeks in the laboratory, Dr. Selden explained.

The automated, focused sequencing system is divided into purification, amplification, Sanger sequencing, separation, and detection steps. “We’re focused now on integrating the individual microfluidic components into a single system.”

Initially, nucleic acids are purified and concentrated to relatively small volumes of 10 to 100 microliters and subjected to highly multiplexed amplification by PCR. “The amplified regions are then subjected to Sanger sequencing, after which Genebench™ separation and detection technology is employed,” Dr. Selden explained. A single sample is interrogated for a large set of biothreats, and multiple loci are sequenced per agent.

Plasmon-Enhanced Systems

Les Baillie, Ph.D., professor of microbiology at Welsh School of Pharmacy, is developing assays to detect nanogram levels of anthrax biomarkers in human blood within 30 seconds. The assay is designed for use by first responders—police, fire, and EMTs—working with crude samples, Dr. Baillie said.

The assays are based on microwave-accelerated metal-enhanced fluorescence (MAMEF), pioneered by Chris Geddes, Ph.D., a professor at the University of Maryland, who has determined that fluorescent probes work better in close proximity to silver surfaces. In fact, many different metals can be used, and 1,000-fold enhancement is possible, Dr. Baillie said. Therefore, nucleic acid amplification isn’t necessary for the detection of genome sequences.

Anthrax spores are highly resistant and can remain in the environment for long periods of time. The researchers recently reported that DNA from Bacillus anthracis spores was detected within a minute in the ng/µL concentration range using low-power focused microwave heating. With this technology, “the microwaves break the resistant spores open in about 30 seconds and also speed up biological recognition,” Dr. Baillie said. Results “are as specific as can be,” correctly distinguishing between B. anthracis and B. cereus, a nonvirulent close relative.

Dr. Baillie is working with the U.K. Ministry of Defence and the U.S. Department of Defense. In addition, he has a close collaboration with Dr. Geddes, who founded The Institute of Fluorescence and also Plasmonix, which will commercialize metal-enhanced fluorescence (MEF) technology.

According to Dr. Geddes, MEF technology has numerous applications outside of biodetection. It can increase the sensitivity and speed of many diagnostic and biological assays, he reported, including two tests in development that work within 20 seconds—one for enzymes that are elevated as a result of myocardial infarction and another for chlamydia.

PCR Alternative

An alternative to PCR that can provide mobile, on-site testing by first responders has been developed by TwistDx.

Based on recombinase polymerase amplification (RPA) isothermal nucleic acid amplification and detection technology, it can detect pathogens, transgenes, and genetic markers within five to 10 minutes, making diagnosis possible within about 30 minutes of sample collection, according to the company.

Compared to PCR, RPA “operates without a need for thermocycling and at a wide temperature range,” explained Helen Kent, operations manager.

“RPA may achieve additional speed by the fact that reiterative primer-elongation events do not require completion of synthesis from the opposing side, as in PCR. In this way, amplification combines both a reiterative linear round of replication and strand displacement with geometric-phase aspects driven by reconversion of single-strands to duplexes.”

Additionally, RPA synthesizes defined points in sample DNA, so amplification occurs only if the target region is present, she explained. “RPA can amplify to detectable levels, at optimal temperature, in only six minutes from single-target levels.

“The entire reaction system is stable as a dried formulation and can be transported safely without refrigeration,” according to TwistDx. Other benefits include the ability to function on crude samples and low, constant-temperature operation.

For example, the optimal operation temperature is 37ºC, but the process will work—albeit more slowly—at a typical ambient temperature of 25ºC. Therefore, it may be possible to develop a simple, inexpensive disposable test system for use in cold environments or field conditions, Kent said.

Sensitivity is such that RPA can detect single copies of DNA and 10 copies of RNA. That is sufficient, TwistDx says, to operate at the single molecule level in the presence of hundreds of nanograms of unrelated complex genomic DNA, like that from plants and mammals. Several different targets may be detected with a single test because multiple detection primers can be combined in one tube.

DVDs Meet Biosensing

At Philips Research, Reinhold Wimberger-Friedl and his team have developed a compact fluorescence reader based on confocal scanning with submicron resolution and single fluorophore sensitivity, which could be taken into the field or used in small labs.

“Since the main optical components are derived from optical storage technology, the instrument can be as compact and low cost as a portable DVD player,” he said. And, it is robust and easy to use, he added.

“As a demonstration, we have developed a simple sandwich assay for C-reactive protein with printed spots on a plastic substrate and fluorescently labeled antibodies. We were able to detect 100 fM without further assay optimization,” Dr. Wimberger-Friedl said. The device is well-suited for real-time hybridization studies because it “allows detection of bound targets at the surface with low interference from background noise originating from the solution above the surface.”

Essentially, the system is an x-y scanner with moving optics and a static object or substrate. It operates in reflective mode through the substrate. “In combination with confocal detection, background signals are suppressed, which allows real-time hybridization measurements. The auto-focus capability makes the system robust,” Dr. Wimberger-Friedl reported.

The device minimizes background fluorescence and provides a low-cost instrument for the laboratory and for less experienced users, according to Dr. Wimberger-Friedl. The technology is based upon Philips’ pioneering DVD work, adapted to biosensing.

“We set ourselves the goal of achieving single binding event resolution so that we could do digital biosensing much like the digital optical recording. This would give us the highest sensitivity and dynamic range,” Dr. Wimberger-Friedl said.