Biological and chemical threats are not confined to the aftermath of the terrorist attacks of 2001. A pound of sodium cyanide, with the potential to injure or kill hundreds of people, was found in a downtown Denver hotel last August, as party luminaries were gathering to prepare for the Democratic National Convention. In the U.S., the Department of Defense (DOD) is determined to detect such threats early enough to mitigate their effects.
New research aimed at the detection of biological or chemical agents is resulting in a handful of precise, rapid detection and identification devices that can be used in the field or lab. Several of these advances were outlined at CHI’s “Systems Integration in Biodefense” conference held recently.
The goal, from the perspective of the DOD, is to deploy state-of-the-art early- warning systems that feature integrated sample preparation and target analysis in a multiplexed, field-deployable format. Multiuse applications are preferred.
At the conference, SAIC and Thermal Gradient described a high-volume aerosol-detection system for biological threats that may be permanently deployed in areas that are considered probable targets. The detection system, designed under contract with the Department of Homeland Security, routinely samples the air, performing automatic identification when possible threats are detected.
As a trigger, the device would use a UV laser-induced fluorescence sensor that extracts particulates from the air. If the particles resembled weaponized pathogens, their DNA would be amplified using a high-speed, multiplex PCR system that can perform amplification within five minutes. The substances are identified using multi-color fluorescence readouts, and results are matched against a list of likely threats provided by the DOD. The time from the initial trigger to threat confirmation is about 10 minutes, and a whiff of another scent such as perfume won’t trigger it.
Thermal Gradient provides the PCR component of the device, using microfluidics in a flow-through silicon chip to perform multiplex PCR amplification of threat-specific genomic nucleic acid, according to Joel Grover, Ph.D., CEO. The chip itself measures 14 x 20 x 2 mm and requires only a few watts of power. It can multiplex about 20 agents simultaneously.
“What we’ve designed provides eight logs of PCR amplification in five minutes or less, which is much faster than anything available today,” said Dr. Grover. Such speed is possible because only the sample is heated, he explained. In contrast, traditional PCR methods heat and cool, not only the sample but the vessel as well thus increasing the heating and cooling needs and lengthening the heating and cooling times.
Canary in a Coalmine
Innovative Biosensors (IBI) has developed an integrated biological aerosol collector and identification system to detect and identify biological threats as part of a building’s sophisticated security system. The device is divided into the BioFlash™ analytic module and the BioDisc™ collection module.
“The sample is collected directly into the BioDisc,” explained Jeanette Simpson, Ph.D., senior scientist, thus eliminating the need for sample preparation.
The pathogen is detected by a Canary™ B cell—a genetically modified B lymphocyte in which the native antibodies have been replaced with antibodies against the pathogen of choice. Identification is made via a bioluminescent immunoassay. Biological threats are detected and identified within three minutes, she elaborated. Sample size ranges from about 50 to 250 agent-containing particles per liter of air, thereby meeting the most stringent levels required by the U.S. government.
“The device is designed primarily for building security,” Dr. Simpson added, and may be installed in the air-handling system either for triggered monitoring or timed interval monitoring. “The device itself can pull in 480 liters of air per minute and is optimized to detect particles between 1 and 10 microns in size. It currently can detect the agents on the DOD’s target list and has the capability of identifying modified biological agents through their conserved sites.”
The device was commercialized from work advanced at MIT’s Lincoln Labs. “IBI is the sole licensee,” added Dr. Simpson. “In addition to its aerosol-detection capabilities, IBI has developed a liquid-based assay system and is working on a hand-held instrument.”
Akonni Biosystems discussed its integrated detection solutions, as well as its sample-prep products. The firm will be going to market later this year with its TruTip™ technology for DNA purification. Currently, the device is undergoing field testing.
Moving from sample collection to 10-fold concentrated and inhibitor-free DNA takes about four minutes, according to Kevin Banks, Ph.D., marketing advisor and founder. “There’s a large demand to clean-up raw samples quickly, without labor-intensive steps like centrifuging.”
Dr. Banks attributes the speed of TruTip to a DNA-modified matrix that fits into the pipette tip. Lysis, binding, washing, and elution all occur in the pipette tip, which handles up to 1.5 mL of fluid, “so you can work easily with large, field-based samples,” he explained.
“As you wash, you remove inhibitors that affect back-end PCR-amplification processes. We’ve even amplified DNA isolated from bacteria living from dirt.” The TruTip concentration effect results in 1.5 mL of raw sample being reduced to about 50 microliters of PCR-ready target in four minutes.
The TruArray™ integrated detection solution is a low-density microarray system that is capable of detecting as few as 500 copies of DNA per sample. Once the DNA is purified and amplified, it is hybridized onto as many as 200 probes for multiplex analysis using the TruArray chip. All the combined steps take about two hours. Then the optical reader analyzes the result. “The ability to interrogate across multiple genes and bacterial species at the same time gives the system enormous power,” Dr. Banks elaborated.
In addition to its biothreat detection capabilities, TruArray assays are also being developed for clinical applications to detect multiple drug-resistant tuberculosis and methicillin-resistant Staphylococcus aureus.
Magnetic Beads Separation
Microchip Biotechnologies is advancing its microfluidic program into the prototype phase. In this phase, the company is focusing on sample preparation as the most commercially viable component of its early development program. “There’s an urgent need for universal sample preparation,” pointed out Joanne Horn, Ph.D., senior staff scientist.
“The BeadStorm™ platform uses magnetic beads to purify cellular targets from such complex matrices as blood or aerosols. The process is straightforward,” she added. The purified target sample is lysed mechanically with technology that emerged from work at the Keck Graduate Institute of Applied Life Sciences.
“Then, released DNA is captured on a second set of magnetic beads, and the concentrated sample is magnetically separated and then washed.” The advantage of magnetic beads, Dr. Horn pointed out, is that they purify and concentrate the target in one step, leaving the output as DNA that has been selectively separated, concentrated, and purified for specific targets.
The platform is engineered to interface with several different downstream analytic platforms, according to Dr. Horn, and possibly with an upstream raw sample collector such as an aerosol sampler. To do so, a modular design is vital.
“The heart of the BeadStorm system is the plastic cube, a 1 inch (2.5 cm) disposable processing device that processes samples using an interfaced, bonded microfluidic chip,” she added.
The system has been used successfully in forensics work involving DNA extraction and purification from buccal swabs, and is being expanded to include an upstream immunomagnetic separation in the workflow. “That process was optimized in previous work.”
The sample-prep technology could be used in the field to monitor military field operations and personnel, as well as testing air, water, and food.
The technology can process four multiplexed samples in about 25 minutes. Microchip Biotechnologies plans to expand that to eight samples. The benefits of this platform include simplified operation and increased accuracy through automation, use of fewer reagents, and multiplexing, according to the company.
IQuum is using its Liat™ system, based upon its lab-in-a-tube technology, to develop a field-deployable identification and confirmation system that is fast, accurate and inexpensive. This integrated system is fully automated, from accepting raw sample through real-time PCR and agent identification. As such, is it designed for first responders who may lack specialized training.
The system includes the disposable, flexible, Liat Tube, with prepackaged reagents, a stand-alone analyzer, an integrated sample processor with an embedded computer for network connectivity, and automation control and readout display.
Assays are performed by actuators that compress the tube to selectively release reagents, move the sample from one segment of the tube to another, and control reaction condition by adding magnetic beads or other substances. A 50 microliter rapid PCR can perform 30 samples in about seven minutes. The complete test for an infectious agent, from sample to results, takes about an hour, the company reports.
Also at the biodefense meeting, Samsung Advanced Institute of Technology (SAIT) discussed its fully integrated, pathogen specific DNA extraction device.
The tool is capable of extracting cDNA from whole blood on a CD, using centrifugal microfluidics on the CD platform itself, lysing samples via laser irradiation, separating particles with target-specific antibodies conjugated to magnetic beads, and moving those samples using Ferrowax microvalves.
The entire process, from plasma separation through DNA extraction was completed within 12 minutes and, except for manually loading 100 microliters of whole blood, was completely automated, the firm asserts.
The study extracted hepatitis B and E. coli, with results comparable to those acquired using usual benchtop methods, according to SAIT.