We have developed and tested a fully autonomous pathogen detection system (APDS) capable of continuously monitoring the environment for airborne biological threat agents. The system is designed to provide early warning to civilians in the event of a terrorist attack. The final APDS will be completely automated, offering aerosol sampling, in-line sample preparation fluidics, multiplexed detection and identification immunoassays, and orthogonal, multiplexed PCR (nucleic acid) amplification and detection. The system performance (current capabilities include aerosol collection, multiplexed immunoassays, sample archiving, data reporting, and alarming) was evaluated in a field test conducted in a Biosafety Level 3 facility, where the system was challenged with, and detected, a series of aerosolized releases containing two live, virulent biological threat agents (Bacillus anthracis and Yersinia pestis). Results presented here represent the first autonomous, simultaneous measurement of these agents.

Continuous monitoring of the environment for infectious diseases and related biowarfare agents requires the implementation of practical cost-effective methodologies that are highly sensitive and specific. One compatible method employed in clinical diagnostics is real-time polymerase chain reaction (PCR) analysis. The utility of this technique for environmental monitoring is limited, however, by the utilization of single-use consumables in commercial PCR instruments. This greatly increases mechanical complexity, because sophisticated robotic mechanisms must replenish the disposable elements. An alternative strategy develops an autonomous monitoring system consisting of reusable modules that readily interface with fluidic circuitry in a flow-through scheme. The reduced complexity should increase reliability while decreasing operating costs. In this report, we describe a reusable, flow-through PCR module that functions as one component in such a system. This module was rigorously evaluated with Bacillus anthracis genomic DNA and demonstrated high repeatability, sensitivity, and efficiency, with no evidence of sample-to-sample carryover.

A field-deployable instrument has been developed to detect low-level 2,4-dinitrotoluene (2,4-DNT) vapors. The system is based on previously developed artificial nose technology and employs an array of sensory materials attached to the distal tips of an optical fiber bundle. Both semiselective and nonspecific, cross-reactive sensors were employed. Each sensor within the array responds differentially to vapor exposure so the array's fluorescence response patterns are unique for each analyte. The instrument is computationally "trained" to discriminate target response patterns from nontarget and background environments. This detection system has been applied to detect 2,4-DNT, an analyte commonly detected on the soil surface above buried 2,4,6-trinitrotoluene (TNT) land mines, in spiked soil and aqueous and ground samples. The system has been characterized and demonstrated the ability to detect 120 ppb 2,4-DNT vapor in blind (unknown) humidified samples during a supervised field test.