Dean R. Hadley

Environmental Monitoring for Biological Threat Agents Using the Autonomous Pathogen Detection System with Multiplexed Polymerase Chain Reaction

We have developed and field-tested a now operational civilian biodefense capability that continuously monitors the air in high-risk locations for biological threat agents. This stand-alone instrument, called the Autonomous Pathogen Detection System (APDS), collects and selectively concentrates particles from the air into liquid samples and analyzes the samples using multiplexed PCR amplification coupled with microsphere array detection. During laboratory testing, we evaluated the APDS instruments response to Bacillus anthracis and Yersinia pestis by spiking the liquid sample stream with viable spores and cells, bead-beaten lysates, and purified DNA extracts. APDS results were also compared to a manual real-time PCR method. Field data acquired during 74 days of continuous operation at a mass-transit subway station are presented to demonstrate the specificity and reliability of the APDS. The U.S. Department of Homeland Security recently selected the APDS reported herein as the first autonomous detector component of their BioWatch antiterrorism program. This sophisticated field-deployed surveillance capability now generates actionable data in one-tenth the time of manual filter collection and analysis.

Handheld advanced nucleic acid analyzer

There is a growing need for portable, lightweight, battery operated instruments capable of detecting and identifying bio-warfare and bio-terrorism agents in the field. To address this need, we have developed a handheld PCR instrument. LLNLs advanced thermal cycling technology and expertise with portable, field tested biological instrumentation, combined with the development of real-time, fluorescence based PCR assays, has enabled the development of a very portable, versatile, power efficient PCR instrument with a simplified operating system designed for use by first responders. The heart of the instrument is the sample module, which incorporates the advanced silicon thermal cycler developed at LLNL.

Miniaturized detection system for handheld PCR assays

We have developed and delivered a four chamber, battery powered, handheld instrument referred to as the HANAA which monitors the polymerase chain reaction (PCR) process using a TaqMan based fluorescence assay. The detection system differs form standard configurations in two essential ways. First, the size is miniaturized, with a combined cycling and optics plug-in module for a duplex assay begin about the size of a small box of matches. Second, the detection/analysis system is designed to call a positive sample in real time.

Advantages Afforded by Miniaturization and Integration of DNA Analysis Instrumentation

Miniaturization of chemical reaction chambers, along with the integration with low-cost detection components for real-time product quantification allows for significant improvements in instrumentation. Bulk chemical reactions can benefit from increased control at the microscopic level, according to the general volumetric reaction formula:

Functional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device

Q\, = \,1/U\int_{qr} {dU}

A miniature analytical instrument for nucleic acids based on micromachined silicon reaction chambers.

where, Q is the volumetric reaction rate (moles/time · volume), U is the volume, and qris the “point” reaction rate at a “microscopic” volume unit. This equation is only valid if everything is uniform at a microscopic scale, that is, the reaction is working equally at all points. This uniformity is difficult to maintain, especially in reactions that have: 1) multiple co-reactants, 2) narrow and uniform condition (i.e., (temperature and pH) requirements, 3) macromolecular biological components, such as enzymes, and 4) diffusion limiting conditions. Typically, bulk bioreactors require significant effort to maintain such uniformity and often are difficult to scale up as a result. Arrays of miniaturized reactors with individual control can replace the less effective bulk systems, provide better uniformity, and therefore increase productivity.

Rapid pathogen detection using a microchip PCR array instrument

Two Ricor K506B Stirling cycle cryocoolcrs with H-10 control electronics were used on the Clementine spacecraft, one cooling a 256 × 256 InSb infrared detector for the near-infrared (NIR) camera and the other cooling a 128 × 128 HgCdTe focal plane array on the longwave infrared (LWIR) camera. This is the first use of these Ricor Stirling cryocoolers in a space environment. This mission has demonstrated the use of these Clementine lightweight imaging sensors in the demanding environment of space, and and has space-qualified this Ricor cryocooler. Moreover, nearly the entire 38 million square kilometers of the Moon’s surface was mapped in 11 spectral bands, six in the near-infrared, during the 71 days of lunar mapping. The more than 1.6 million digital images collected are providing mineral typing scientific data to the international civilian scientific community.