M. DONLON AND J. JACKMAN

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DARPA Integrated Chemical and BiologicalDetection System

Mildred Donlon and Joany Jackman

he threat of attack on military and civilian targets with chemical and biologicalweapons is a growing national concern. The Defense Advanced Research ProjectsAgency (DARPA) is developing technologies for detecting these materials in thenatural environment. While several technologies show promise as broadband detectors,there is no “silver bullet” that detects all chemical and biological materials at therequisite levels of sensitivity and specificity. DARPA is developing a systems approachwhereby several different advanced detection schemes (based on different physicalphenomena) are being integrated into a chemical and biological detection suite.(Keywords: Biochemical detectors, Chemical detectors, Lateral flow devices, Miniatur-ized mass spectrometer, RNA chip technology.)

INTRODUCTIONThe number of toxic or infectious agents that could

be used in a military or civilian environment is quitelarge. These materials range from simple inorganic ororganic chemicals to complex bioengineered microor-ganisms (Fig. 1). At one end of the spectrum are vapor-phase nerve agents that will kill nearly instantly. In themiddle are biochemicals such as neuroactive peptidesthat have low vapor pressure but have extremely dis-ruptive effects on human life. At the other end areinfectious bacterial agents that will kill in a dose of onlyone organism but with a longer onset time. Developinga detection system that combines the speed, sensitivity,and specificity necessary for environmental detectionof these highly diverse classes of materials, in a packagethat is field portable, is a daunting task.

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DETECTION OF THE THREATThe approach taken by the Defense Advanced Re-

search Projects Agency (DARPA) is to view the entirespectrum of the threat as a collection of chemicals thatincrease in size and complexity on going from left toright in Fig. 1. When seen from this view, the threatspectrum can be logically divided into a part that ismade up of a single molecular compound (chemicalagents, emerging chemical agents, bioregulators, andtoxins) and a part that is a complex and ever-evolvingmixture of biochemicals (microbes and engineeredmicrobes). Historically, the volatile chemical agentshave been detected using vapor-phase detection equip-ment such as ion mobility spectrometers, surfaceacoustic wave devices, or gas-sampling mass spectrom-eters. Similarly, the microbes have been detected using

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Figure 1. Chemical and biological threats (SEB = staphylococcal enterotoxin B).

Chemicalagents

Emergingchemicalagents

Bioregulators Toxins Microbes Engineeredmicrobes

Nerve agentsBlister agentsBlood agents

Toxic chemicals

Aerosols

Neuropeptides

Psychoactive compounds

RicinSEBSaxitoxinMycotoxin

BacteriaVirusesSpores

Genetically manipulated

Micro- encapsulates

Vapor Solid

10–3 g/person 10–12 g/person“Toxicity”

Detection requirementFast Slower

antibody-based technology with optical reporters.Inspection of the threat spectrum reveals significantdeficiencies in these technologies. The vapor-sensingequipment is completely ineffective for the larger,nonvolatile mid-spectrum and microbial threats. Thecurrent antibody-based technologies also do not effec-tively cover the mid-spectrum threat and suffer fromspeed and sensitivity problems. DARPA has developedand fostered a systems engineering approach to coverthis gap in field detection capability.

DARPA’s approach is to invest in the best chemicaland biochemical broadband detection capability that isbeing developed and to augment this capability withthe latest highly specific and sensitive identificationtools for microbial threats. In particular, DARPA hasfunded the development of a miniaturized time-of-flight (TOF) mass spectrometer system at APL as abroadband chemical and biochemical detection tech-nology, the multiplexed upconverting phosphor flowcytometer and lateral flow devices at SRI International,and the RNA chip technology at Argonne NationalLaboratory for identification of specific microbes.These systems have been chosen to match up with thetemporal response and sensitivity requirements of thethreat spectrum. For instance, it is imperative thatmany of the chemical agents be detected in near–realtime because of the speed with which they act onhumans. On the other hand, infective biological agentsmay take many hours before the onset of debilitationor death. However, early recognition of a potentialmicrobial attack (even without specific identification)is crucial because it enables simple prophylactic mea-sures (such as filtering masks) to be taken to dramat-ically reduce the casualty rate.

Critical for effective medical treatment after an in-cident is verification of the putative attack and specificidentification of the biological agent, albeit with a

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DARPA INTEGRATED CHEMICAL/BIOLOGICAL DETECTION SYSTEM

somewhat relaxed time constraint.The system funded by DARPA usesthe rapid detection capability ofthe mass spectrometer to identifychemical and mid-spectrum threatsand to detect and characterize themicrobial threat within a few min-utes. Using information providedby the mass spectrometer as a trig-ger, the flow devices and RNA chiptechnologies are used to providemore specific identification of thethreat over a period ranging fromseveral minutes to a few hours. Theessential battlefield requirementsof protection through early warn-ing of troops and identification ofthe threat to enhance the outcome

of medical countermeasures are incorporated in thissystem.

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MINIATURIZED TIME-OF-FLIGHTMASS SPECTROMETER

The mass spectrometer is the most powerful labora-tory analytical tool for analyzing a broad spectrum ofchemical and biological materials. Mass spectrometry isa technique for determining the masses of moleculesand specific fragmentation products formed followingvaporization and ionization. Detailed analysis of themass distribution of the molecule and its fragmentsleads to identification of the molecule. These molecularmeasurements can be done at the attomole (10–18 mole)level of material using specialized laboratory-basedinstruments. The combination of specific molecularidentification and extreme sensitivity is what makesmass spectrometry such a powerful tool. While suchcapability has existed in the laboratory for many years,the development of a small, portable mass spectrometerfor potential field detection of chemical and biologicalsubstances is described here.

APL is developing a miniaturized TOF mass spec-trometer system for automated environmental detec-tion. This system combines a continuously vapor-sam-pling electron impact ionization method for detectingsmall chemical substances and a laser desorption/ioniza-tion method for detecting higher-weight mid-spectrumand microbial materials. For brevity’s sake, we describeonly the biological detection scheme here.

For the particulate threat, a trigger will initiate anaerosol sampler to sort, concentrate, and collect mate-rial onto a movable tape sample substrate. The sampleis moved into the vacuum chamber of the mass spec-trometer for analysis. The application of pulsed lightfrom a laser onto the target creates sample ions. The

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instrument uses matrix-assisted laser desorption/ioniza-tion (MALDI). With this technique, biomolecules aslarge as 300,000 Da can be ionized and desorbed intactinto the gas phase for mass analysis.

The basis for MALDI is the interaction of a pulsedlaser beam with a laser-absorbing matrix material intowhich analyte molecules are dispersed. Pulsed laserenergy is absorbed by the matrix and transferred to theanalyte, causing it to be ionized and desorbed into thegas phase. In the process, the analyte chemically inter-acts with fragment ions of the matrix, forming molec-ular adduct ions. The performance of the mass spec-trometer system is outstanding for chemical andbiochemical materials, including the mid-spectrumthreats described. These biochemicals can be detectedwhen presented individually to the mass spectrometerand can be determined in more complex mixtures.

The ultimate in complex mixtures is exhibited inmicrobes such as viruses and bacteria. The TOF systemis proving useful for initial classification of these ma-terials through the building of databases of spectralinformation and development of the interpretive soft-ware tools for using the database information for detec-tion. The database methods and data are being devel-oped at the University of Maryland, College Park, andthe U.S. Army Research Institute of Infectious Diseasesusing a chemotaxonomic approach.

The chemotaxonomic method recognizes that allmaterials of biological origin are composed of commonbuilding blocks that include inorganic chemicals,small organic molecules, carbohydrates, amino acids,and nucleic acids. These building blocks are linked inprecise fashion to form all manner of biological ma-terials. These larger macromolecules are the primesignatures that can be used to identify materials ofinterest. Chemical markers that are characteristic ofbroad classes of microorganisms as well as markers thatare specific for specific pathogens are being developed.These markers can be quantified extremely rapidlyusing the techniques of modern mass spectrometry.

The polar lipids present in bacterial cell (and severalother) membranes are classically a useful chemotaxo-nomic biomarker. These biochemicals are also attrac-tive (and useful) as mass spectral biomarkers. Thephospholipid biomarker libraries include both molec-ular ion fingerprints and polar head group analysesderived from fragment ions.

While polar lipids are present in bacterial cells andenveloped viruses, they are absent in most viruses andall proteinaceous toxins. A more universal class ofbiomarker is based on peptides and proteins. The useof this type of biomarker is complicated by the largenumber (several thousand) of proteins present in atypical cell. However, proteins are being identified thatare characteristic of specific bacteria and viruses.

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Finally, the genetic pattern (DNA and RNA) ofbacteria and viruses is an absolutely characteristicbiomarker. However, current mass spectrometer tech-nology does not allow long-chain DNA and RNA tobe measured. DARPA is funding approaches thatuse chemical and enzymatic production of smallerfragments (oligonucleotides) for direct fingerprinting ofviruses. Nucleic acid analysis using mass spectrometryis currently best accomplished by first enzymaticallydegrading the nucleic acid, using probes immobilizedon a surface that capture specific sequences, and thenmeasuring the mass distribution on the surface. Withproperly designed probes, a mass spectrometer can ef-ficiently generate genetic information.

As pointed out, chemical building blocks making upbacteria and viruses can be measured by mass spectrom-etry. In addition, the broadband mass spectrometer canbe used to measure contaminants (spent media, encap-sulation materials, and other cofactors of weaponiza-tion) that are present in weaponized biological mate-rials. However, the information produced is quitecomplex, requires significant interpretation, and relieson the building of an accurate database. Although thistechnique is improving steadily, it does not yet providethe definitive identification (down to the strain level)often necessary for biological detection and diseasetreatment. To fill this gap in rapid identification ofbiological agents, DARPA has invested in a handheldflow-through biodetector, an advanced flow cytometerwith upconverting phosphor reporters, and RNA chiptechnology with both fluorescent and mass spectrom-etry readouts.

FLOW CYTOMETRY WITHUPCONVERTING PHOSPHORS

DARPA has also invested in the development of aminiaturized flow cytometer and a handheld bioassayincorporating a novel reporter system for multiplexeddetection and identification of biological agents. Theflow cytometer system is based on the flow of a fluidstream carrying a low density of biological particlespast an illuminating laser. As the individual particlesenter the interaction area of the laser, light is scatteredinto several different optical detectors. Informationabout the size and shape of the particles can be gath-ered from the spectral and angular distribution of thescattered light. The flow cytometer is a well-estab-lished tool in the clinical laboratory for identifying andcounting large cells such as white and red blood cells.More recently, the technique has been applied to muchsmaller bacterial cells in the microbiological researchlaboratory. The laboratory systems are quite sensitiveand provide useful information about the identity ofthe biological materials, particularly when antibody

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probes are used. More recently, the sensitivity andspecificity of the flow cytometer have been enhancedthrough the use of specific dyes and fluorescently la-beled antibodies. Furthermore, with the judicious se-lection of labeled reporters, multiplexing of multipleagents can be obtained.

While this tool has become increasingly useful in thelaboratory, it has not transitioned to a field-portabledevice because of the requirement for a high-powerlaser system and the weak signals from the reportersystems used. SRI has been developing a unique classof reporter materials based on mixed rare-earth oxides.These man-made oxides have remarkable optical prop-erties unlike anything in nature. Their electronic struc-ture is such that they exhibit an efficient near-infrared,two-photon absorption followed by a visible-light flu-orescent emission. Thus, these materials can be used asreporter molecules based on absorption of near-infraredlight that is readily obtained using small, light, andinexpensive semiconductor lasers. Furthermore, thevisible emission line characteristic of natural materialsis not observed, so the emission can be detected with-out background effects, thus dramatically reducing sam-ple noise and enhancing signal sensitivity. The mixedoxide upconverting phosphor materials have been sur-face modified with various binding sites and are avail-able with a variable mixture of rare earths so that theemission spectrum of the probe can be chosen for agiven analyte. The net effect is a highly efficientmultiplexing of the flow cytometer.

The upconverting phosphor materials have pavedthe way for miniaturization of the flow cytometer andhave led to the enhancement of antibody-based hand-held assays. Because the reporter probe can be excitedwith small semiconductor lasers, the entire system cannow move out of the laboratory and into the field.Although the technological advances have broughtflow cytometry into the arena of fieldable resources, thelack of available specific probes has limited their bat-tlefield use. Focus on the flow-based systems for fielduse has shifted to the production of highly specificprobes for identifying microbes. Antibodies that iden-tify a particular microbe within a genus or species areabundant for some microbes such as Staphylococcus.Until very recently, there were no antibody-basedprobes that could distinguish potentially pathogenicstrains of Bacillus and Yersinia from nonvirulent species.This is a critical point for environmental detectors,which take samples in areas that may be naturally richin these genus members.

Novel approaches to synthesize small, specific-rec-ognition molecules that could be used to identify theseorganisms are under way at the University of Alabama,Birmingham (UAB) and Utah State University(USU). UAB and USU have undertaken peptide

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phage display against pathogenic strains of Bacillus andother pathogenic microbes to screen for small, specificmolecules. While UAB is using a combinatorial libraryof random peptides, USU is combining this approachwith the analysis of specific bacterial receptors as ameans of discovering useful probes for identification.Other researchers at UAB have recently reported thefirst antibodies that can speciate the Bacillus genus.

RNA CHIP TECHNOLOGYThe most characteristic molecules for identifying an

organism are the genetic materials DNA and RNA.Researchers from the Englehardt Institute in Moscow,working at Argonne National Laboratory, have devel-oped a tool for rapidly identifying microorganisms usinga three-dimensional gel pad chip. The target moiety intheir investigation is ribosomal RNA (rRNA). SincerRNA is present in many thousands of copies per cell,the selection of this target obviates the need for signalamplification using PCR (polymerase chain reaction)-based methods. Therefore, analysis of natively ampli-fied RNA dramatically speeds the analysis compared toother nucleic acid identification approaches.

In the evolutionary history of life, the RNA mole-cule and the ribosome structure were among the firstobjects developed. In particular, the 16S fraction ofrRNA is intriguing to study as it appears to be one ofthe genetic elements that encodes the road map ofevolutionary progression and divergence from simple tocomplex organisms. The 16S rRNA consists of 1500nucleotides in a characteristic structure. As life hasevolved, this molecule has evolved with it. All livingcreatures, from bacteria to humans, have rRNA mate-rials within their cells. In the RNA structure there arehighly conserved regions along with regions that showconsiderable variability. By judicious selection of RNAcomplementary probes, regions of the 16S rRNA canbe used to detect and phylogenetically distinguish or-ganisms using the RNA chip.

Scientists at Argonne National Laboratory havedeveloped a two-dimensional array of complementarynucleic acid probes immobilized within a 10 3 60 mmgel pad. The gel pads act as very small test tubes forcarrying out the annealing reactions between the com-plementary probes and the rRNA oligonucleotides.Detection is done by breaking open the cells of interest,cleaving the rRNA, allowing the resulting oligonucle-otides to flow over the chip, and measuring the fluo-rescence of the individual pads using a fluorescentmicroscope. From a knowledge of the complementarysequence, the sequence of a particular section of RNAcan be determined and the organism can be identified.This type of chip can distinguish organisms that differin only a single base pair at the target site.

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The first application of this technology has been thedevelopment of a “Bacillus Chip.” The genus Bacillusis important for biological detection applications be-cause it includes the organism responsible for anthrax.However, the surface structure of spores of the genusBacillus is such that highly specific antibodies for an-thrax are not readily available. The Bacillus Chip is atechnology that allows anthrax to be specifically differ-entiated from closely related organisms. One caveat ofthe rRNA-based chip is that it is of little use in detect-ing viruses, which do not contain an amplified targetsuch as rRNA, and proteinaceous toxins, which maycontain only small variable amounts of contaminatingnucleic acids. These caveats may be overcome by am-plifying the signal through PCR-based methods or byenhancing the detection of signals that are present inlow amounts using other technologies such as the massspectrometer for readout.

An alternative reporting scheme is also being devel-oped in a collaboration between APL and ArgonneNational Laboratory. The approach uses a mass spec-trometer to read out the bound oligonucleotide. Itpresents the opportunity for enhanced sensitivity andfurther multiplexing as a result of the ability of the massspectrometer to determine multiple bindings to a par-ticular sequence. This hybrid approach embodies themass spectrometer with the specificity that is missingin the straightforward chemotaxonomy that is currentlybeing pursued.

SYSTEM INTEGRATIONThe detection of the entire range of toxic and in-

fective threats is not currently possible with one typeof field-portable instrumentation. The best approach tothis broadband detection is the TOF mass spectrometer,which can rapidly and sensitively gather informationon the entire spectrum of the threat. For chemical and

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biochemical threats, the spectrometer can both detectand identify threats because of the ability to measuremolecular mass and fragmentation patterns. For com-plex microorganisms, the information that the massspectrometer currently provides leads to classificationof the threat and identification of co-factors of weap-onization. The mass spectrometer does not at this pointhave the database and algorithms for identification ofthe threat. It is imperative in the short term to havefieldable technologies that address the entire threatspectrum.

The upconverting phosphor flow cytometer and theRNA chip are being integrated into a detection suitethat provides a hierarchical approach to detection,classification, and identification. In this scheme, mul-tiple sensors based on different technologies are used.In this way, countermeasures against one technologyshould prove ineffective against the others.

For the scenario of troop protection in the field, thedetection system would work as follows: The systemwould be stationed in the field, and intelligent particlecounters (e.g., the Lincoln Laboratory Biological Aero-sol Warning System) would continuously monitor theair for a rise in specific bioparticle counts. When sucha rise occurs (or when another trigger is alerted), theaerosol collectors on the system would be activated.Samples would be collected on the mass spectrometersampling tape, and analysis of the threat would com-mence. On the basis of the information from the massspectrometer about the nature of the threat (chemical,biochemical, or microbial), the sample collected for theflow cytometer and RNA chip would be analyzed onthose systems. Thus, triggering, detection, classifica-tion, and identification would be rapidly and accuratelyobtained from the integrated DARPA chemical andbiological detection system. As other systems for rapiddetection and identification are developed, they will beincorporated into an enhanced detection system.

THE AUTHORS

MILDRED DONLON has an M.S. degree in radiation biology and biophysicsand a Ph.D. in microbiology and immunology, both from the University ofRochester. She joined DARPA in 1994 as a program manager in biologicalwarfare defense. She has been serving the Department of Defense for 22 years,first as a bench scientist and more recently as a program manager. Her e-mailaddress is mildonlon@darpa.mil.

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JOANY JACKMAN received her Ph.D. from the Program in Cell and MolecularBiology at the University of Vermont in 1990 and then joined the MolecularPharmacology Group at the National Cancer Institute. She joined the faculty ofGeorgetown University School of Medicine in 1994, where she continued toinvestigate the biochemistry and genetics of disease processes. Since 1997, Dr.Jackman has been an IPA with the Special Pathogens Branch of the U.S. ArmyMedical Research Institute of Infectious Diseases. She is involved with thetesting of biosensors and their component parts against live biological threatagents as part of the DARPA Biosensor Program. Her e-mail address isjjackman@ftdetrck-ccmail.army.

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