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.
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.