Pathogens, such as bacteria and viruses, and the toxins produced by some organisms are viewed as potentially easier weapons of mass destruction for terrorist groups and rogue governments to develop, and as such, require particular vigilance in detection and identification on the part of the world community. Spectroscopic methods have been used for many years to amplify the signal provided by primarily nucleic acid–based and antibody-based detection regimes; these continue to be improved and refined, alongside newer attempts to develop spectroscopic techniques that can be used in the absence of specific molecular interactions. For example, mass spectrometry currently is being applied in proteomic-type methods to identify pathogenic bacteria from groups of proteins within them that possess fragments of unique masses; in effect, a pathogen-specific mass signature. Conjugation of gold nanoparticles and fluorescent reporter groups to specific antibodies or nucleic acids that reliably bind to unique partners, characteristic of a particular pathogen or toxin, allows a variety of spectroscopic techniques to amplify the signal arising from ever smaller numbers of fluorophores or associating nanoparticles. Among these spectroscopic methods are a number exploiting surface plasmon resonance, two-photon Rayleigh scattering, surface-enhanced Raman spectroscopy, and probe-dependent fluorescence quenching. Electrochemical methods for detecting specific reporter–substrate interactions also are being exploited for their sensitivity. Here, the recent research literature is reviewed to identify representative advances in the use of spectroscopy to improve detection capabilities for pathogens and toxins potentially used as bioweapons, with respect to specificity, sensitivity, rapidity, and portability to the field.

There is growing concern today among scientists, policymakers, and the general public that pathogenic microbes, such as bacteria and viruses, and toxins generated by a variety of organisms might be converted into biological weapons by terrorist groups or rogue governments (1). The primary motivating factors for this concern about the possibility of "weaponizing" a pathogen or toxin are the ready availability of the relevant microorganisms, the limited need for resources to effect the transformation of a pathogen into a weapon (all of which would be available to interested governments and organizations for a price most could manage), and the ease of transmission of the biological agent into and among the target population (2). Though some commentators have suggested that the interest among radical groups and governments in these weapons is considerably less than generally supposed (3,4), the ease of deployment of such a bioagent dictates an aggressive defense (5). Making pathogens and organisms that synthesize toxins unavailable, or severely restricting their dissemination to prevent their misuse, is simply not feasible: even 

, the causative bacterium in anthrax, is widespread in the soil where livestock are raised (6). Clearly, the key to protecting human populations from biothreats used maliciously lies in rapid detection and identification of the dangerous agent, both before and during a bioterror attack. Improvements in detecting and identifying such bioagents most likely will arise from advances in detecting smaller amounts of the relevant material more rapidly and reliably, minimizing sample preparation before evaluation, and making detection and identification technology portable to any environment where needed. 

Spectroscopy is a key tool in improving methods for detecting and identifying bioterror agents, as most key molecules constituting such agents are of sufficient complexity that they have identifiable spectroscopic features and give strong signals. Currently, most instrumental techniques for generally detecting and identifying microbes, toxins, and biothreat agents constituting a smaller group within that larger classification scheme rely on one of two methods for recognizing a specific molecular entity:  

Antibodies which bind specifically to a defining molecular group or set of groups on the surface of the agent, or 

Nucleic acids of specific sequences that pair complementarily to particular sequences in the nucleic-acid population (typically the genome) of the agent organism.

Both of these specifically binding entities are highly engineered by nature to recognize their targets with extremely high specificity and affinity, even among much more numerous competing molecules of similar structure.  

Detecting the formation of the specific complex, especially at low concentrations, is where spectroscopic methods traditionally have made their greatest contributions to this specific detection and identification effort. Attachment of a fluorescent label or a uniquely labeled nanoparticle to the antibody or reporter nucleic acid makes the antibody's or nucleic acid's behavior accessible to a variety of spectroscopic methods, but more recently, experimenters have been developing techniques to exploit the spectroscopically active groups on the target molecules themselves, such as the toxin of interest or a cell-surface or virus-surface carbohydrate or protein. Others have adapted established reporter-based techniques to more compact and portable instrumentation, or have broadened the applicability of a method to more complex and less purified samples, permitting more rapid determinations with less sample preparation. 

Fluorescent Detection of Antibody-Based Reporter Groups

Because of the high specificity of a single antibody for its epitope (the group on the target antigen to which the antibody binds), a wide variety of methods for identifying and quantifying specific molecules have been based on antibodies over the last several decades. Many of these methods can be extended to viruses and bacteria (or other types of cells, such as fungi or cancer cells) if the molecule of interest is located on the surface of the larger entity. Warner and colleagues (7) have attached a quantum-dot reporter group to antibodies directed against the A-type botulinum toxin (produced by the bacterium 

, the causative organism in the food-poisoning syndrome botulism), a protein of common concern as a potential bioterrorism agent. These investigators used the quantum dot in the traditional "sandwich"-type immunoassay (Figure 1), in which a "capture" antibody is bound to a solid surface and then used to hold the antigen (botulinum toxin) while other molecules are washed away. A second antibody, for detection, which also binds to the antigen of interest and bears the fluorescent reporter group, is incubated then with the solid support and forms an antibody "sandwich" with the antigen in between. After excess detection antibody is washed away, the quantum dot fluoresces (at 655 nm) under activation by light at the excitation wavelength (365 nm). The quantum dot gives both greater sensitivity, allowing detection in the low picomolar range, and more stable emission signal, because of the lack of photobleaching seen with an earlier reporter group.  

Figure 1: The quantum-dot-fluorescence, "sandwich"-type immunoassay of Warner and colleagues (7). The toxin molecule being assayed (heart-shaped, dark green) is "sandwiched" between the capture (dark blue and green) and detection (light blue and red) antibodies. Those capture antibodies not binding a toxin molecule do not retain any detection antibodies after the final wash is performed, so the quantum dot (yellow diamond) fluoresces in proportion to the bound detection antibody, and thus in proportion to the bound toxin, when excited by laser light of the appropriate wavelength. The capture antibody is bound to the solid support, anchoring the entire complex throughout the washes and the spectroscopic analysis, by a short linker (purple oval). 

Fluorescent Detection of DNA Amplified by Polymerase Chain Reaction

Since the development of the polymerase chain reaction (PCR) technique two decades ago for the amplification of very small amounts of DNA, visualization and quantification of the DNA produced has depended most commonly on the fluorescence of a reporter group indicating the quantity of the desired DNA present. In recent years, real-time PCR has used instruments with built-in fluorescent optics and continually more clever ways to employ fluorescent reporter groups to reflect as accurately as possible the presence and quantity of the desired DNA molecule. Kutyavin (8) recently reported a technique he describes as "snake" (because of the shape of the hybridized DNA intermediate formed), in which an oligonucleotide probe designed to basepair exclusively with an interior segment of the target DNA is degraded by the polymerase's 5'→3'-nuclease activity, separating the attached fluorophore from its neighboring quenching agent and allowing the fluorophore to fluoresce in proportion to its free concentration (Figure 2). The "snake" modification improves the signal-to-noise ratio of the fluorescence monitoring by keeping the fluorescent probe able to bind the target DNA independently of the portion of the DNA being synthesized, rather than becoming incorporated into the synthesized strand. The author did not test the method specifically on DNA segments associated with bioterrorism threats, but the method is applicable, in principle, to any pathogenic organism, such as the bacteria giving rise to anthrax, bubonic plague, staphylococcal infections, Legionnaire's disease, and a host of other illnesses. The primary advantage of PCR in detecting microorganisms is that it requires little sample and almost no sample preparation (the small sample size allows most contaminants simply to be diluted out in constituting the reaction).  

Figure 2: "Snake" real-time polymerase chain reaction assay, monitored by fluorescence. In the work of Kutyavin (8), a primer used for amplification (complementary to the light blue 3'-end of the DNA strand shown here) is synthesized with a 5'-extension containing the same sequence as that to which the "flap" (light blue segment) pairs. This allows the "snake" secondary structure to form (black and light blue DNA strand). The fluorescent probe DNA, bearing a quenching group (yellow square) at its 3' end, basepairs with the segment immediately downstream of where the flap hybridizes, which allows the polymerase's (dark green, heart-shaped entity) 5'â3' nuclease activity to degrade the probe. This degradation of the probe releases the nucleotide bearing the fluorescent label (green circle) from the proximity of the quencher, permitting a fluorescence signal in quantitative agreement with the concentration of DNA synthesized (and, therefore, bearing the flap sequence). 

Use of Nanoparticles to Detect Pathogenic Bacteria

Two-photon Rayleigh scattering: Singh and colleagues (9) used the two-photon Rayleigh scattering properties of gold nanorods (~100 nm in length, aspect ratio 2.7) to develop a sensitive method of detecting specific bacteria at low concentrations. The investigators conjugated a monoclonal antibody to the nanorods, directed against a cell-surface antigen unique to 

 O157:H7, a strain often implicated in food poisoning outbreaks. Even at low concentrations of bacterial cells (50 viable cells per milliliter), enough nanorods aggregate to provide a reproducibly detectable two-photon scattering intensity, distinguishable from the background associated with free nanorod-conjugated antibodies or another strain of 

 at significantly higher concentration. The spectral technique owes its aggregation-dependent sensitivity to the surface-plasmon excitation properties of the gold nanorods; one photon arising from the longitudinal electronic disturbance, and the other from the transverse effect, with the large aspect ratio contributing the substantial difference in wavelength between the two photons. A significant advantage of this type of technique is that it operates on intact bacterial cells, eliminating the need for disruption of the cells to make the antibody target accessible, as is the case with many nucleic-acid-based probes. It also avoids the need for additional antibodies of other specificities for detection, as in the widespread "sandwich" assays (the target molecule or particle "sandwiched" between the capture antibody, which binds it to the solid surface, and the detection antibody, which carries the signal-amplifying label, as described above and in Figure 1). 

Direct Identification of Bioterror Agents by MS

During the past couple of decades, mass spectrometry (MS) has evolved to handle continually larger molecules, including proteins and complex carbohydrates. In the interest of pathogen identification, typically that of bacteria, some investigators have turned to profiling whole cells using the method, and matching the extremely complicated spectra that result, to libraries of such spectra for known microorganisms. Christner and colleagues (10) have recently used matrix-assisted laser desorption–ionization, time-of-flight (MALDI-TOF) MS to "fingerprint" a variety of blood-borne bacterial pathogens and test the limitations of the technique. They were able to identify the organism correctly in 87% of the 277 cases they attempted, with the failures largely arising from excessively small numbers of cells in those samples. The investigators also determined that Gram-positive bacteria, such as 

, were more likely to fail to be identified than Gram-negative bacteria, such as 

 species. The development of reliable methods for identifying intact cells will be a significant step forward in bioterror-agent detection, as much less sample preparation will be required than for the analysis of individual components of those cells. MS is particularly well suited to this application because of the extremely high resolution and lack of overlapping signals it exhibits in contrast to other spectroscopic techniques. 

Although whole-cell, MS-based analysis of a microorganism permits identification of that organism, the individual peaks in the spectrum often are not correlated to the identities of the individual molecules giving rise to them. A common use of MS to characterize pathogens is to fractionate the purified pathogen into individual molecular components, typically proteins, and then to identify each component separately using the spectral technique. Kong and colleagues (11) recently separated the constituent proteins of infectious bronchitis virus (IBV), a coronavirus infecting chickens (the virus responsible for the sudden acute respiratory syndrome [SARS] epidemic in Asia in 2003 was also a coronavirus), by two-dimensional gel electrophoresis, and then identified the individual proteins by digesting them with trypsin and subjecting them to MS. Additionally, the investigators found a large number of host-cell proteins packaged in the virus particles, identified by spectrum-matching to a library of proteins. Although the technique used here may shed light on the structure of the virus and vulnerabilities that can be exploited in confronting it clinically, the requirement to purify the virus and isolate it in significant quantities is a drawback to applying this method to identifying the virus in clinical samples. Viruses typically are present clinically in extremely low quantities, and require antibody- or PCR-based detection methods. However, the technique described by Kong and colleagues would be appropriate for identifying an unknown substance confiscated or discovered by authorities and thought to be a purified virus intended for use in a bioterrorism attack. 

Direct Identification of Bioterror Agents by Vibrational Spectroscopy

Vibrational techniques, such as infrared and Raman spectroscopies, incorporate an enormous amount of structural information from their subject molecules. However, each suffers from a disadvantage: Infrared signals are swamped by the spectral contributions of water, which occurs in large quantities in biological samples, while the Raman technique shows extremely weak signals because of the small number of photons scattered. Driskell and colleagues (12) recently applied surface-enhanced Raman scattering (SERS) to the identification of a variety of rotaviruses, in which the isolated virus was deposited on an array of silver nanorods, such that laser excitation of the mobile electrons in the silver atoms enhanced the Raman peaks because of the virus particles by several orders of magnitude. The spectra for the individual virus species then were characterized by principal-component analysis, to obtain a reproducible spectral profile for each species against which unknown samples could be matched. Because of the intense signal enhancement resulting from the metal surface, the virus preparation did not require concentration, and cell lysates could be diluted somewhat before application to the silver substrate. Thus the sensitivity of the technique likely is sufficient to analyze bodily fluid samples from infected patients, but perhaps not yet high enough to detect virus reliably in those who are still presymptomatic. 

Use of Surface Plasmon Resonance to Characterize Toxin Binding

Flagler and colleagues (13) recently used surface plasmon resonance to compare the binding preferences of Shiga toxins 1 and 2, which are often mentioned as potential bioterror agents, toward the cell-surface carbohydrates they exploit to enter cells and exert their toxicities. The results of the surface plasmon resonance binding determination were qualitatively similar to those obtained using an enzyme-linked immunosorbent assay (ELISA — an antibody-based detection method similar to the "sandwich" assay described above and in Figure 1), but the ELISA was more sensitive by a couple of orders of magnitude. The conditions under which the surface plasmon resonance experiment was conducted may be possible to refine further to improve the method's sensitivity. However, it is also possible that the continuous flow of the toxin-containing solution, compared to the lack of agitation in the ELISA, plays a much greater role in destabilizing the complex, and that surface plasmon resonance is simply not the ideal choice for studying this specific toxin–receptor complex. 

Electrochemical Detection of Specific Molecules

Liu and colleagues (14) recently have published work in which they devised an electrochemical sensor for the presence of interferon-γ (gamma) that could, in theory, be modified to detect the presence of a toxic biothreat agent, such as botulinum toxin or ricin. The authors use an aptamer, which is a nucleic-acid molecule that folds into a specific conformation capable of binding a target molecule with substantial specificity. Here they employ a 34-nucleotide DNA segment, that, absent interferon-γ, forms a stem-and-loop structure, bringing the redox-labeled 3'-end in proximity to the 5'-end, which is bonded to a gold surface serving as an electrode (Figure 3). When interferon-γ binds to the aptamer, it disrupts the stem-and-loop structure, drawing the redox label away from the gold surface and impeding electron transfer. The extent of the impedance is quantified by square-wave voltammetry, and the authors determine that it is in proportion to the amount of interferon-γ present and shows high sensitivity (ability to detect, reliably, quantities in the range observed in living organisms).  

Figure 3: Electrochemical detection of interferon-Î³ by aptamer-based electrical impedance. According to Liu and colleagues (14), the binding of the subject molecule, in this case interferon-Î³ (gray oval), disrupts the basepairing (blue and red ladder) between the complementary segments of the aptamer (dark blue, green, and red structure). This draws the redox label (yellow square) out of proximity of the gold surface, the latter serving as an electrode, and the resulting distance (narrow blue line on far right) is too great to support electron transfer. The result is a measurable electrical impedance indicating the concentration of interferon-Î³. The aptamer is linked to the gold surface by a short thiol-bearing group (magenta oval), permitting the subject molecule to be washed away and the redox label to be regenerated. 

It is clear from current research in bioterror-agent detection that fluorescence spectroscopy is still the major tool by which specific target molecules are quantified, and that trapping these molecules is still done, for the most part, using antibodies or amplifying them using PCR. However, because of continually increasing sensitivity in MS, the direct detection of pathogen by-products and whole cells by that class of methods is growing rapidly. Other spectroscopic methods, including those of electrochemistry, are finding utility in detection schemes as they can be applied to the recognition of specific features on target molecules, or conjugated to other molecules that specifically recognize the targets. The methods and examples cited here are not necessarily the most appropriate among the many others, which were not mentioned, but are representative of the current trends toward more specific, sensitive, rapid, and portable means to detect biological threats of a wide variety. 

 (3) Y. Sharan, "The Bioterrorism Threat," in 

Risk Assessment and Risk Communication Strategies in Bioterrorism Preparedness (NATO Security Through Science Series A: Chemistry and Biology)

, M. Green et al., Eds. (Springer, Dordrecht, The Netherlands, 2007), pp. 47–54. 

is with Protein Molecules, Inc., Springfield, Illinois. 

Articles

Article

More-specific, sensitive, rapid, and portable spectroscopic methods detect biological threats.

Advances in Spectroscopy for Detection and Identification of Potential Bioterror Agents

Traditionally, an indication of exposure to chemical warfare nerve agents (CWNAs) is a depression of cholinesterase activity. However, this method is nonspecific for etiology and source and generally cannot differentiate between past and current exposures. We have evaluated the biomonitoring of CWNAs in various biological matrices from guinea pigs using the technique of fluoride ion regeneration followed by GC–MS-MS analysis.

Although the use of chemical warfare nerve agents (CWNA) such as sarin (

-isopropyl methylphosphonofluoridate, military designation GB), cyclosarin (

-cyclohexyl methylphosphonofluoridate, GF), and VX (

-diisopropylaminoethyl methylphosphonothioate) (see Figure 1) is forbidden by the Chemical Weapons Convention, documented cases of the use of these nerve agents exist (1–3). The extreme toxicity of these nerve agents makes retrospective detection of potential CWNA exposure an important issue both for the civilian population and military personnel. When exposure is sufficient, clinical symptoms observed include salivation, miosis, tremors, and death (4). These physiological effects result from inhibition of the cholinesterase enzymes distributed throughout the nervous system with a resultant build up of the neurotransmitter acetylcholine. However, when exposure occurs at a very low level, few symptoms, if any, may be present. Therefore, assessing exposure is done typically by means of biomonitoring, a measurement of the internal dose for biological monitoring of exposure.  

Traditionally, an indication of exposure to nerve agents is a depression of cholinesterase activity, specifically erythrocyte acetylcholinesterase (AChE), which has been shown to correlate with the severity of symptoms of CWNA poisoning at high exposure levels. However, this method is nonspecific for etiology and source and generally cannot differentiate between past or current exposures. Furthermore, because of the large inter- and intra-individual variation of erythrocyte AChE activity, a marginal inhibition is impossible to assess unless pre-exposure values have been determined. We have evaluated an alternate method of biomonitoring. 

The method reported here employs the fluoride reactivation or regeneration approach first described by Polhuijs and colleagues (1), wherein the corresponding phosphonofluoridate (GB or GF) is regenerated from an inhibited phosphonyl moiety after the addition of fluoride ions. For the VX exposures, biomonitoring employs the fluoride-induced reactivated phosphonyl moiety of VX, 

-ethyl methylphosphonofluoridate, or VX-G (5–7), also shown in Figure 1. The VX-G, GB, GF, and deuterated analogs were isolated by means of solid phase extraction followed by analysis using isotope-dilution gas chromatography–tandem mass spectrometry (GC–MS-MS). 

 The animal model of choice for this study was the male Duncan-Hartley guinea pig, weighing 250–350 g (Charles River Labs, Kingston, New York). Whole-body inhalation exposures (GB and GF) were conducted in a 1000-L dynamic airflow Rochester-style inhalation chamber (8). Animals were exposed for 60 min to either 1.6 mg/m

. Before removing the animals, the chamber was purged with air for 10 min after completion of agent exposure. For percutaneous exposures, a 60-μg/kg (0.4 × LD

) 10% VX, 90% isopropanol dosage was applied to the left flank of the animals where their fur had been clipped previously using a 1-μL Hamilton syringe (Hamilton Company, Reno, Nevada). Immediately after agent application, a plastic cap was placed over the exposure site and held in place by double-sided carpet tape. A self-adhesive elastic bandage was wrapped several times around the animal to cover the exposure site, carpet tape, and plastic cap. At 6 h after agent application, the bandage was cut and the carpet tape and plastic cap were removed, exposing the application site. All studies were approved by the U.S. Army Medical Research Institute of Chemical Defense Institutional Animal Care and Use Committee and in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International. Carotid artery blood draws (0.5–1.0 mL) were taken before the start of the exposure and at periodic intervals throughout into BD Vacutainer tubes (BD, Franklin Lakes, New Jersey) containing K

EDTA and stored at 4 °C until analysis. Tissue samples were stored at –70 °C. During the study, the guinea pigs were observed for clinical manifestations of agent toxicity, but none were observed. 

 For the blood assays, the target analyte was extracted using either Sep-Pak C18 or Oasis HLB solid-phase extraction (SPE) cartridges (both from Waters Corp., Milford, Massachusetts) following a pH adjustment to pH 3.5 with a sodium acetate–acetic acid buffer and the addition of potassium fluoride. This induces the formation of the phosphonofluoridate from either protein bound or free agent. A deuterated internal standard also was added before the solid-phase extraction for quantitation. The analytes and internal standards were eluted with ethyl acetate. Tissue sample preparation also employed freeze-fracture pulverization under cryogenic temperatures using a Tissue Cryoprep system and an S-series focused acoustic energy system (Covaris, Woburn, Massachusetts).  

Figure 2: Total ion chromatogram of 200-pg standard. 

 Analyses were performed using an Agilent Technologies (Santa Clara, California) model 6890 gas chromatography (GC) system interfaced to a Waters Micromass Quattro micro GC tandem quadrupole mass spectrometer. GC separations were achieved using a 30 m × 0.25 mm, 0.25-μm 

 Restek Rtx-5MS column (Bellefonte, Pennsylvania). The carrier gas was helium with a flow rate of 1 mL/min. Injections of 1.0 μL were made by autoinjector into a splitless injector port at a temperature of 250 °C. The initial oven temperature of 35 °C was held for 1 min, then ramped at 15 °C/min to 100 °C, and ramped again at 35 °C/min to 280 °C and held for an additional 1 min. Typical elution times are shown in Figure 2.  

Samples were ionized by positive-ion chemical ionization (CI) with ammonia reagent gas. CI source conditions were optimized using Fluoroether E3 tuning compound with methane reagent gas. Mass spectra were obtained at a dwell time of 0.05 s for each transition in the multiple reaction monitoring (MRM) mode. Argon was used as the collision gas at a pressure of ~2 mTorr, with a collision-induced dissociation (CID) energy of 10 eV. The following transitions were monitored for the respective analytes: 

Table I: Precision and accuracy from saline spikes 

 Figure 3 shows the response of the chemical warfare nerve agents to the internal standards as a function of picograms agent injected onto the column (for a 1-μL injection) for a series of calibration standards prepared in ethyl acetate. The data are an average taken over several weeks and show good correlations over two orders of magnitude (VX-G, 

 = 0.9987). The precision (presented as the coefficient of variation or CV) and accuracy (or mean) of the assays were determined by replicate analyses (

= 5) of multiple aliquots of saline spiked at three different concentrations. These concentrations represented a low quality control (LQC), a medium quality control (MQC), and a high quality control (HQC) equivalent to the concentrations of biomarkers on column. The results are summarized in Table I. Recoveries of the biomarkers were measured by spiking blood with either VX, GB, or GF at the equivalent of 0.1 nmol of agent per gram of blood and assaying for the respective analyte. Results varied from 60% to 80%.  

Figure 4: Concentration of GB in various tissues as a function of time post-exposure. 

 Figure 4 shows the concentrations of regenerated sarin (GB) determined in various tissues as a function of time following the 60-min inhalation exposure. As expected, once the nerve agent enters the systemic circulation, it is distributed throughout the tissues of the body. Each value represents the average of six or seven individual determinations. As the data indicate, there is a definite decrease in the concentration of GB with time. Based on additional concentration/time measurements (not shown), all four biological matrices exhibit typical first-order elimination kinetics of the form 

, where half-lives for regenerated sarin were estimated as blood, 3.4 h; lung, 22 h; liver, 11 h; and kidney, 14 h. Figure 5 shows the same plot for the concentrations of regenerated cyclosarin (GF) as a function of time following a similar 60-min inhalation exposure. In general, the data are similar to the GB exposure with comparable half-lives for regenerated GF.  

Figure 5: Concentration of GF in various tissues as a function of time post-exposure. 

Figure 6 shows the concentrations of VX-G analog in various tissues as a function of time following a percutaneous exposure to VX. These data clearly show the effect of the dermal exposure route. Although blood levels remain essentially unchanged, VX-G concentrations in the three target organs actually increase with time. As the data indicate, there is a significant amount of VX-G recovered in the skin at the site of exposure even 24 h later. A spontaneous release of this agent from bound sites in the skin with a systemic circulatory uptake might account for the observed target organ increases. This mode of action could impact postexposure decontamination and treatment efforts.  

Figure 6: Concentration of VX-G in various tissues as a function of time post-exposure. 

The authors wish to acknowledge Drs. Christopher Whalley and Lucille Lumley for their technical assistance as Principal Investigators, and all the members of the Analytical Toxicology Branch for their help with the regeneration assays. This research was supported by the Defense Threat Reduction Agency. DTRA neither endorses nor recommends the use of any products, techniques, or materials mentioned or pictured here. 

(1) M. Polhuijs, J.P. Langenberg, and H.P. Benschop, 

(2) M. Minami, D.M. Hui, M. Katsumata, H. Inagaki, and C.A. Boulet, 

(3) H. Tsuchihashi, M. Katagi, M. Nishikawa, and M. Tatsuno, 

(5) E.M. Jakubowski, L.S. Heykamp, H.D. Durst, and S.A. Thomson, 

(6) C.E.A.M. Degenhardt, K. Pleijsier, M.J. van der Schans, J.P. Langenberg, K.E. Preston, M.I. Solano, V.L. Maggio, and J.R. Barr, 

(7) J.M. McGuire, J.T. Taylor, C.E. Byers, E.M. Jakubowski, and S.A. Thomson, 

(8) C.E. Whalley, J.M. McGuire, D.B. Miller, E.M. Jakubowski, R.J. Mioduszewski, S.A. Thomson, L.A. Lumley, J.H. McDonough, and T.-M.A. Shih, 

Jeffrey M. McGuire, Edward M. Jakubowski, Jr., and Sandra A. Thomson 

are with the US Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland. 

A fluoride-regeneration approach enables biomonitoring of chemical warfare nerve agents.

Monitoring of Biological Matrices by GC–MS-MS for Chemical Warfare Nerve Agent Detection

Portable Raman analyzers have great potential for identifying explosive materials associated with improvised explosive devices. However, most commercial analyzers employ 785-nm lasers that can generate fluorescence interference, limiting identification capabilities, and possibly cause permanent eye damage because of the use of high powers and open laser beams. Here we compare the Raman spectra obtained for TNT and RDX using 785-, 976-, 1064-, and 1550-nm lasers. The latter is of special interest, as it falls within the retina-safe spectral range.

Close to 50% of all United States casualties in Afghanistan and Iraq are due to improvised explosive devices (IEDs)(1,2). The United States Army has been investigating the use of portable Raman analyzers to potentially identify such devices, as well as the raw materials used to make them. The choice of Raman spectroscopy is primarily due to its ability to identify virtually any chemical based on its unique spectrum, and the fact that the sample can be measured noncontact without preparation using a point-and-shoot probe. The choice of excitation wavelength plays a critical role in obtaining quality spectra. Nearly all portable Raman analyzers employ 785-nm laser excitation, which can generate fluorescence in samples and diminish, even eliminate, identification (3,4). Of specific concern are TNT, a secondary explosive often used in IEDs, and RDX, a primary explosive and major component in C4 and Semtex, both of which exhibit fluoresce in some samples. Furthermore, highly colored glass containers may also generate fluorescence interference. Longer wavelength lasers, such as 1064 nm, can be used to avoid fluorescence (5), but with a concomitant loss of sensitivity, due to the fact that the Raman signal intensity decreases as the wavelength increases to the fourth power (that is, the υ

In addition to fluorescence interference and sensitivity, eye safety is also a concern, especially since the end-users are not likely to have extensive experience using high powered lasers (for example, 500 mW). Laser wavelengths at 785 and 1064 nm are transmitted through the cornea and focused on the retina, increasing the power density by several orders of magnitude, and even a few milliwatts of power can cause permanent eye damage (7). Furthermore, all of the current commercial portable Raman analyzers use laser powers exceeding the maximum permissible exposure (MPE) limit of 1–5 mW stated by the American National Standards Institute (ANSI) (7), necessitating the use of sample enclosures or laser glasses by all personnel in the vicinity of the measurement.  

In an effort to address this latter concern, we have developed a portable Raman spectrometer that employs 1550-nm laser excitation. This wavelength falls within the 1400–2000 nm "eye-safe" range (preferably referred to as retina-safe [7]), so-called because the least amount of damage to the eye occurs in this spectral region (8). In contrast to wavelengths below 1400 nm, the retina-safe wavelengths are not focused by the eye, but are absorbed by the cornea, aqueous and vitreous humor (Figure 1). The cornea can handle a 10-s exposure to 1 W/cm

  of a 1550-nm continuous wave laser for 50% of a population before thermal damage (9). Furthermore, the cornea can repair itself in several days. For the retina-safe region ANSI sets the MPE at one tenth this value, 100 mW/cm

Figure 1: Illustration of the human eye showing the focus of visible radiation on the retina and absorption of 1550 nm radiation by the cornea, aqueous, and vitreous humor. Damage to the retina is permanent, while damage to the cornea is temporary, but should still be avoided. 

To our knowledge there has been only one Raman spectroscopy publication using a retina-safe laser (10). This is primarily due the υ

  dependence of Raman scattering. For example, a Raman vibrational mode at 1000 cm

  will be 21.5 times as intense at 785 nm compared to 1550 nm. In addition, the spectral response of quality detectors is limited to a 0–2000 cm

  Raman range at this excitation wavelength. Here, the trade-offs in obtaining Raman spectra for TNT and RDX using 785-, 960-, 1064-, and 1550-nm excitation are presented in the context of eye safety. 

Approximately 0.1 mg each of 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX) were obtained from the United States Army. In all cases the samples, TNT as grains and RDX as a powder, were placed into 2-mL glass vials, which were in turn placed in the Fourier-transform (FT) Raman spectrometer sample compartments for measurements. All four FT-Raman spectrometers used to perform all measurements were designed and built by Real-Time Analyzers (Middletown, Connecticut). The interferometers for all four spectrometers were identical in design and optical components (11,12). A 300-mW 785-nm laser (Process Instruments, Salt Lake City, Utah) was used with a silicon avalanche photodiode detector (PerkinElmer, Stanford, Connecticut), while a 1-W 976-nm laser (in house design), and 1064- and 1550-nm lasers (Keopsys, Lannion, France) were all used with InGaAs detectors (Judson, Montgomeryville, Pennsylvania). The fiber-optics and sample probe optical designs were identical for all systems, except for the Raman line filter used to remove spurious radiation (for example, silicon Raman scattering) and the long pass Raman edge filter used to remove the laser Rayleigh scattering (Semrock, Rochester, New York and Omega Optical, Brattleboro, Vermont). These filters were matched to the appropriate laser wavelength. The probes were mounted under light tight covers to ensure eye safety and eliminate ambient light interference. 

The eye safety of the four systems is defined first. According to ANSI the following laser power upper limits must be met to qualify as Class 1 and be considered eye-safe (7): 0.56 mW for 785, ~1 mW for 976, 1.9 mW for 1064 nm, and 9.6 mW for 1550 nm. Of course, virtually all laboratory Raman spectrometers employ enclosures to eliminate potential exposure to the eye, and thereby qualify as Class 1. However, the fiber-optic probes of the systems used in this study could also be handheld without a cover (as they are in many commercial portable Raman analyzers), in which case they would all be Class 3 for laser powers between 10 and 500 mW, and must be considered potentially harmful requiring the use of laser goggles. For open-beam designs, such as handheld probes, ANSI defines a nominal ocular hazard distance (NOHD) for a laser emitting from a focusing lens (7):  

 is the lens focal length (here the probe lens, 18 mm), 

 is the laser beam diameter at the lens (5.4 mm, based on the laser fiber-optic collimator), Φ is the laser power (300 mW for 785 nm and 500 mW for the other three wavelengths) and MPE is the maximum permissible exposure limit for a given wavelength calculated using ANSI tables (Table I).  The eye-safe distance is the NOHD plus the lens focal length. Notice that the eye-safe distance is quite far at nearly 2.5 ft for 785-nm laser excitation, compared to only 4 in. for 1550 nm. This suggests that for the former all personnel in the vicinity of an open-beam portable analyzer should be wearing laser safety glasses, whereas only the operator needs safety glasses for the latter.  Needless to say, no portable Raman analyzer using an open-beam probe is truly eye-safe, and in the case of the 1550-nm system, damage to the cornea can occur if a person's eye is within the 4-in. distance. It is also worth noting that the MPE for skin is similarly 100 mW/cm

, and the heat of the laser can be readily felt! Finally, only the 785-nm beam is visible to the unaided eye, while the other wavelengths are invisible, offsetting some of the improved safety associated with the longer wavelengths.  

Table I: Maximum permissible exposures and nominal ocular hazard distances for the listed excitation wavelengths using 500 mW 

The ability to measure the Raman spectra of TNT and RDX is discussed in order of increasing laser excitation wavelength. As previously stated, a number of explosives fluoresce when excited by 785-nm laser excitation (4), which is true for these two explosives. The extent of the fluorescence interference is often dependent on the purity and age of the samples, and in the case of TNT, the fluorescence contribution is less for newly purified TNT than that shown in Figure 2a, but more for older, real-world samples. It is also worth noting that the responsivity of the Si detector drops considerably at about 1950 nm, corresponding to a 2500 cm

  Raman shift using 785-nm excitation, and consequently the CH vibrational modes are barely discernable. Even though the υ

  dependence of Raman scattering coupled with the increased responsivity for Si versus InGaAs detectors favors this laser wavelength, the primary TNT peaks are only modestly discernable above the fluorescence. Nevertheless, it should be stated that the quality of the spectrum (signal-to-noise ratio, S/N) is quite good for a 10-s acquisition time using 300 mW of laser power. But it must be kept in mind that variations in the amount of fluorescence can challenge even the best spectral search and match software used to identify an unknown. In general the same results are obtained for RDX using 785-nm excitation (Figure 2b); the spectrum contains a substantial amount of fluorescence, but the primary peaks are discernable, and a high quality spectrum is obtained in a short time with modest laser power.  

Figure 2: Raman spectra of (a) TNT and (b) RDX measured using 785-nm laser excitation (normalized intensity scale). Conditions: 300 mW, 10 s acquisition time, and 8 cm-1 resolution. 

Although the Raman spectra of both explosives do not contain fluorescence using 976-nm laser excitation, a second interferent appears. Two broad features several hundred wavenumbers wide and centered at 500 and 900 cm

  are attributed to luminescence generated in the glass sample vials (Figure 3). Although the primary RDX peaks are clearly identifiable for these explosives, other explosives (for example, ammonium perchlorate [4]) that only contain unique Raman peaks in this low wavenumber spectral range may be difficult to identify. Additional tradeoffs occur using this excitation wavelength. Because an InGaAs detector is used, the entire spectrum is available for unknown identification using spectral matching, but the overall sensitivity has dropped significantly, and the same quality spectra obtained using 785-nm excitation requires a 1-min acquisition time and 500 mW of laser power.  

Figure 3: Raman spectra of (a) TNT and (b) RDX measured using 976-nm laser excitation (normalized intensity scale). Conditions: 500 mW, 1-min acquisition time, and 8 cm-1 resolution. 

No fluorescence is observed in the Raman spectra of either explosive using 1064-nm excitation (Figure 4). The lack of any spectral interference coupled with the availability of the entire spectrum and a reasonable S/N, even for a 10-s acquisition time, allows for highly successful identification of unknown chemicals using spectral search and match software, as well as discrimination against similar chemicals.  

Figure 4: Raman spectra of (a) TNT and (b) RDX measured using 1064-nm laser excitation (normalized intensity scale). Conditions: 500 mW, 10-s acquisition time, and 8 cm-1 resolution. 

Excitation using 1550 nm also does not generate fluorescence or glass interference in the Raman spectra for either explosive, but the spectral quality is severely compromised even using 500 mW of laser power and 10-min acquisition times (Figure 5). This is not surprising, since the υ

  dependence of Raman scattering predicts the dominant modes for TNT and RDX, the NO

  and the C-N-C ring stretch for RDX at 885 cm

, respectively, to be 16% and 18% of their respective intensities using 1550-nm laser excitation compared to 1064-nm excitation. However, the measured peak intensities are only half this intensity. This is attributed to less efficient optics used in the 1550-nm system, and in particular the fiber-optics coupled to the probe (12). The spectral noise is also higher even after the long acquisition time. This is attributed to a higher noise equivalent power for the extended range InGaAs detector. This excitation wavelength also limits the available spectral range, as the responsivity for this detector approaches zero at 2200 nm, which corresponds to a 1900 cm

  Raman shift, and consequently the CH stretching region is not detected.  

Figure 5: Raman spectra of (a) TNT and (b) RDX measured using 1550-nm laser excitation (normalized intensity scale). Conditions: 500 mW, 10-min acquisition time, and 8 cm-1 resolution. (* indicates laser artifact.) 

Here we compared the Raman spectra obtained using 785-, 976-, 1064-, and 1550-nm laser excitation for two important explosives, TNT and RDX. The data suggest that 1064-nm excitation provides the best quality spectra for identifying explosives: no interference, full spectrum, and reasonable S/N in 10 s. We also demonstrated that Raman spectra for these two explosives could be obtained using a 1550-nm laser emitting in the retina-safe region. The primary advantage for performing measurements using this wavelength laser is added eye safety since only temporary damage to the cornea is expected versus permanent damage to the retina at the shorter wavelengths. Another advantage is that the chance of fluorescence interference is extremely remote. However, using this wavelength has several disadvantages. First, the signal intensities are 5–10 times less compared to 1064-nm laser excitation due to the υ

  dependence of Raman scattering and losses in the optics. Second, there is increased noise associated with the extended range InGaAs detector. Third, the spectral region is limited by the InGaAs detector response to below ~2000 cm

. In spite of these deficiencies, identification of explosives may be possible if high laser powers and long acquisition times are acceptable. 

The authors are grateful for funding from the United States Army and the United Kingdom Ministry of Defence. 

 (1) C. Wilson, Congress. Res Ser., RS 22330 (2007). 

 (3) I.R. Lewis, N.W. Daniel, Jr., N.C. Chaffin, P.R. Griffiths, and M.W. Tungol, 

 (4) R.L. Green, M.D. Hargreaves, and C.D. Brown, 

Homeland Security supplement to Spectroscopy 

 (5) I.R. Lewis, N.W. Daniel, Jr., and P.R. Griffiths, 

 (Academic Press, San Diego, California, 1994). 

 (7) American National Standards Institute, ANSI Z 136.1 -2007, Laser Institute of America, Orlando, FL (2007). 

 (9) J.L. Lambert, C.C. Pelletier, and M. Borchert, 

 (10) K.A. Lynn, G. McNay, D.A. Eustace, N.C. Shand, and W.E. Smith, 

 (12) C. Brouillette, H. Huang, W. Smith, and S. Farquharson, 

Michael Donahue, Hermes Huang, Carl Brouillette, Wayne Smith, and Stuart Farquharson

 are with Real-Time Analyzers, Inc., Middletown, Connecticut.

A portable Raman analyzer with laser excitation at 1550 nm provides "eye-safe" explosives detection.

Detecting Explosives by Portable Raman Analyzers: A Comparison of 785-, 976-, 1064-, and 1550-nm (Retina-Safe) Laser Excitation

Raman spectroscopy is a powerful analytical technique for detecting a wide range of chemicals in any state (solid, liquid, or gas phase) and is widely used in laboratories for chemical identification. Raman spectra resulting from the vibrational modes of a molecule consist of well-defined sharp spectral lines that are unique to the molecule. This property makes Raman spectroscopy useful as a "fingerprint" technique for the identification of various chemicals, including polymorphs. Recently, Raman spectroscopy is emerging as a standoff technique, using gated ICCD detectors and pulsed lasers, with the capability of identifying chemicals in the daytime from distances ranging from 1 to over 100 m and with a fast detection time of a few seconds. In this article we describe the detection capabilities of a compact standoff Raman system developed at the University of Hawaii (Honolulu, Hawaii) for standoff detection of hazardous chemicals and chemicals used in the synthesis of homemade explosives (HME). The compact standoff Raman system uses a regular 85-mm Nikon camera lens as collection optics and can detect various chemicals from a 50-m distance in daytime with 1–10 s integration time. A slightly bigger system using an 8-in. telescope (203 mm) is shown here to detect chemicals from a 120-m distance using only single laser pulse excitation. These systems are suitable for the real-time detection of various chemicals and would be useful for homeland security and environment monitoring applications.

The recent rise in worldwide terrorist activities has sparked a vast need for standoff technologies capable of detecting hazardous chemicals and explosives at sufficiently safe ranges to avoid and neutralize threats from chemical devices. Standoff Raman spectroscopy can detect several hazardous chemicals from a safe distance without the need to make contact with the sample sealed inside glass and plastic bottles. The ability to distinguish water from colorless flammable organic liquids from remote distances during the daytime would be promising for screening water bottles and various liquid containers at airports. Suspicious chemicals and their residues could also be analyzed on site through their Raman spectra. The fast response time and ability to perform chemical analysis on site during the day and night makes the remote Raman system an attractive instrument for homeland security applications. The Raman spectra representing the vibrational modes of a molecule produce results with a high confidence level and very low incidence of false alarms. The spectral features are unique even for various polymorphs of the same chemical. Raman spectra can detect a wide range of chemicals that are of interest from a public safety point of view, such as explosives (TNT, RDX, C4, TATB, HMX, PETN, various nitrates, chlorates, perchlorates, peroxides, and so forth), hazardous materials (e.g., benzene, naphthalene, acetone, various acids, gasoline, and other flammable materials), toxic gases, and various chemical vapors (1). 

At the University of Hawaii (Honolulu, Hawaii), we have been developing a portable remote Raman system suitable for detection of various chemicals, minerals, and explosives. Portable remote Raman instruments previously developed at the University of Hawaii using 5- and 8-in. telescopes have shown the ability to detect various chemicals and minerals from distances of up to 125 m under bright daytime conditions with a short integration time (2–4). Recently, we have significantly reduced the size of our remote Raman system to develop a compact system that could be used to screen for hazardous chemicals at checkpoints and airports. In this article, we show that good quality Raman spectra of various potentially harmful chemicals such as ammonium nitrate, potassium perchlorate, acetone, isopropyl alcohol, nitrobenzene, and various organic and inorganic chemicals could easily be measured from remote distances up to 50 m with a compact remote Raman system utilizing only a regular 85-mm Nikon camera lens as collection optics and with 10 s of integration time. The remote Raman system also uses a small Raman spectrograph developed at the University of Hawaii (5,6).  

The schematic of a pulsed time-resolved remote Raman system is shown in Figure 1. A frequency-doubled mini Nd:YAG pulsed laser source (model Ultra CFR, Quantel Laser, Quantel USA, Bozeman, Montana, 532 nm, 30 mJ/pulse, 20 Hz, pulse width 8 ns), is used in an oblique geometry to excite the target located at a remote distance. A 10× beam expander is used after the laser to control the laser spot size at the target. The laser beam is aligned with the field of view of the Nikon camera lens using a folding mirror. A 2-in. diameter 532-nm notch filter is used in front of the 85-mm Nikon camera lens to block off the Rayleigh scattered light. The scattered light generated by the target is collected by the camera lens, which then focuses it onto a 100-μm slit of the compact spectrograph equipped with a gated thermoelectrically cooled intensified charge-coupled device (ICCD) detector.  

Figure 1: Schematic diagram of compact time-resolved remote Raman system using an 85-mm camera lens as collection optics and compact Raman spectrograph developed at the University of Hawaii. 

Figure 2 shows a photograph of the compact remote Raman system mounted on a pan and tilt stage, which is then mounted on a portable trolley.  The system is assembled on an optical breadboard with mounting screws separated by 1-in. spacing. The photograph depicts the size of the overall system. Further miniaturization of the remote Raman system by replacing the ICCD detector with a mini ICCD detector is under development. 

Figure 2: Photo of compact remote Raman system mounted on a portable trolley for outdoor testing. The system components are mounted on a breadboard with mounting screws separated by 1-in. spacing. 

Samples of various chemicals in liquid state (water, acetone, ethanol, 2-propanol, benzene, nitrobenzene, and ethyl benzene) and powder form (ammonium nitrate, potassium perchlorate, and potassium nitrate) were contained in glass vials 2.54 cm (diameter) × 5 cm (height) and investigated through the closed glass vial. All samples were analytical grade chemicals obtained from Sigma Aldrich (St. Louis, Missouri) or Thermo Fisher Scientific (Waltham, Massachusetts). A photo of the portable remote Raman system during the 50-m test is shown in Figure 3.  

Figure 3: Photograph of the remote Raman system mounted on a portable trolley during 50-m testing. 

The remote Raman spectra from a distance of 50 m during the daytime with 10 s of integration time of ammonium nitrate (NH

) are shown in Figure 4. All major Raman bands necessary for positive identification of these chemicals are clearly observed. These materials are often used in the production of homemade explosive materials. In the spectrum of NH

  is attributed to the symmetric stretching (ν

  are, respectively, the in-plane bending (ν

  ion, and the lattice (translational and rotational) vibrations of the NO

 by the presence of a broad Raman band in the high-frequency region near 3137 cm

, which is due to symmetric stretching vibration of the NH

  ion. The asymmetric stretching mode of the ammonium ion gives a Raman band near 3225 cm

, which contributes to the broadness of the Raman band in the high-frequency region. In the spectrum of KClO

  to the first overtone of the symmetric bending (ν

  ion. The signal intensity of the latter is enhanced by the Fermi-resonance effect. Fermi resonance happens when the vibrational frequency of an overtone mode is in the vicinity of a symmetric fundamental mode (Raman frequency of the ν

, attributed to the antisymmetric bending (ν

, attributed to the antisymmetric stretching (ν

Figure 4: Remote Raman spectra of ammonium nitrate, potassium nitrate, and potassium per- chlorate from a distance of 50 m with a 10-s integration time. All spectra with 532-nm excitation, 30 mJ/pulse, 20 Hz, 50 ns gate width, as measured. Spectra have been shifted vertically for clarity. 

Raman spectroscopy is very sensitive to chemical composition and structure. This is because Raman spectra are representations of the Raman active vibrational modes of a molecule. When a molecule is altered either by adding extra atoms or replacing atoms with a different element, it changes the vibrational frequencies of various modes. The addition of extra atoms to a molecule also changes the total number of Raman active vibrational modes. This results in a significant change in the appearance of the Raman spectrum of the molecule. The process generates fingerprint Raman patterns that are unique to every chemical. Figure 5 shows the standoff Raman spectra of three very similar chemicals — benzene, ethyl benzene, and nitrobenzene — from a distance of 50 m with detection time of 10 s. Benzene and its derivatives are BTEX compounds and have harmful effects on the central nervous system. The strong aromatic C-H stretching Raman line of benzene appears at 3061 cm

. The strong Raman band corresponding to the symmetric breathing mode of the benzene ring is observed at 992 cm

. This vibrational mode is observed at 1002 and 1003 cm

  in ethyl benzene and nitro benzene, respectively. The effect of the addition of the ethyl group to benzene is easily seen in Figure 5 where it shows a broad Raman band in the CH region at 2933 cm

, which is characteristic of the aliphatic C-H stretching of the ethyl group. The aromatic C-H stretching mode of the benzene ring is observed at 3053 cm

  in ethyl benzene. Similarly, the addition of the nitro group gives a very strong band at 1347 cm

  in nitrobenzene due to symmetric stretching (ν

 functional group and the C-H stretching mode of the benzene ring is shifted to 3079 cm

  in nitrobenzene. Other Raman peaks of medium intensity in the nitrobenzene spectrum are at 853, 1003, 1109, and 1588 cm

 bending, aromatic ring bending, C-N stretching, and aromatic ring stretching, respectively. A number of organic substances that are rich in nitro (NO

) groups are often used in explosive devices, such as nitroglycerine, trinitrotoluene (TNT), and triaminotrinitrobenzene (TATB) and can easily be identified through remote Raman spectroscopy.  

Figure 5: Remote Raman spectra of benzene, ethyl benzene, and nitrobenzene during the daytime from a distance of 50 m with a 10-s integration time. Experimental conditions are the same as in Figure 4. 

Highly volatile and flammable organic compounds are used as solvents and reagents in the chemical and materials industry (for example, acetone, diethyl ether, benzene, methanol, and pyridine). These colorless liquids could be concealed in water bottles and pose significant fire hazards for airlines. Presently, there are restrictions on carrying water and other clear liquids through airport check points, mostly due to the lack of detection capability of screening technologies in distinguishing water from various other hazardous clear liquid chemicals. Figure 6 shows that a standoff Raman system provides distinct identification of water from other flammable organic chemicals. The remote Raman spectra of water, ethanol, 2-propanol, and acetone were obtained through glass vials from a distance of 50 m with a 10-s integration time.  

Figure 6: Remote Raman spectra of water, ethanol, 2-propanol, and acetone through glass vials from a 50-m distance showing that water can be easily identified from organic compounds. Experimental conditions are the same as in Figure 4. 

Water gives very strong Raman signals in the 3100–3600 cm

  spectral region and is easily identified from its broad Raman bands. In the water sample, strong broad Raman bands near 3228 and 3423 cm

) vibrational modes of the water molecule, respectively. In contrast, the C-H bonds in most organic chemicals give very strong sharp Raman peaks in the 2800–3000 cm

  region and make the high-frequency region (>2500 cm

) especially useful in distinguishing water from organic compounds. For example, in the spectrum of 2-propanol, the most intense Raman peaks are from the symmetric and asymmetric stretching vibrations of the methyl (CH

, respectively. The symmetric stretching mode of the CH

. In addition, a very broad, weak band extending from 3100 to 3600 cm

  can be found in various kinds of alcohols, attributed to the hydroxyl (OH) stretching vibrations. The most intense Raman peak of acetone is located at 2923 cm

 vibrations of the methyl groups. Another strong peak at 787 cm

  is attributed to the C-C stretching vibrations. Furthermore, a signature peak from the C=O stretching vibrations can be found in all carbonyl compounds, located between 1600 and 1800 cm

The data presented here show the ability of a portable compact remote Raman system utilizing an 85-mm Nikon camera lens as collection optics to detect various chemicals from a distance of 50 m during the daytime with 10 s of integration time (equivalent to 200 laser pulses excitation). The rapid detection and identification of chemicals could be important in many homeland security applications, such as identifying materials on a conveyor belt or emanating from a fast-moving object. At shorter distances, chemical analysis could be done at a very fast rate using only single laser pulse excitation because the Raman signal is inversely proportional to the square of the target distance. Standoff Raman detection of various chemicals at 10 m distance using single-pulse excitation has been demonstrated earlier by our group using a 5-in telescopic Raman system (7). With a slightly bigger 8-in. telescopic system, single-pulse detection could be done at the much longer range of 120 m as shown in Figure 7. A 100-mJ/pulse energy was used for exciting the targets of ammonium nitrate and potassium perchlorate placed inside a 1-in. glass vial. The figure shows five single-pulse measurements of each target, illustrating good reproducibility and high signal-to-noise detection capability.  

Figure 7: As-measured stand-off spectra of ammonium nitrate and potassium perchlorate powders with a single laser pulse from a 120-m distance using an 8-in. remote Raman system. Spectra measured with single 532-nm excitation, 100 mJ/pulse and with 100-ns gate width. Five measurements for both chemicals are shown to demonstrate the good signal reproducibility of the system. 

A standoff Raman system capable of detecting various chemicals with a very high degree of confidence can be useful for homeland security applications where chemicals sealed inside glass and plastic bottles could be identified from a remote distance in real time without need for handling the targets. Standoff Raman spectroscopy clearly identifies explosive and hazardous chemicals in the daytime using fingerprint vibrational modes unique to each chemical. 

This work was supported in part by grants from NASA and ONR. The authors would like to thank Nancy Hulbirt and May Izumi for their valuable help with figures and editing. 

(1) S.K. Sharma, A.K. Misra, and B. Sharma, 

(2) A.K. Misra, S.K. Sharma, C.H. Chio, P.G. Lucey, and B. Lienert, 

(3) S.K. Sharma, A.K. Misra, P.G. Lucey, S.M. Angel, and C.P. McKay, 

(4) A.K. Misra, S.K. Sharma, P.G. Lucey, R.C.F. Lentz, and C.H. Chio, 

(5) S.K. Sharma, A.K. Misra, S.M. Clegg, J.E. Barefield, R.C. Wiens, and T. Acosta, 

(6) A.K. Misra, S.K. Sharma, D.E. Bates, and T.E. Acosta, 

 76650U, DOI: 10.1117/12.849850, (2010). 

(7) A.K. Misra, S.K. Sharma, and P.G. Lucey, 

Anupam K. Misra, Shiv K. Sharma, Tayro E. Acosta, and David E. Bates 

are with the Hawaii Institute of Geophysics and Planetology, University of Hawaii, Honolulu, Hawaii.

A compact standoff Raman system can be used to detect hazardous chemicals and chemicals used in homemade explosives synthesis.

Detection of Chemicals with Standoff Raman Spectroscopy

The use of multiple optical sensor modalities in a common platform for explosives detection has many advantages over single-sensor design. The combination is particularly useful in a standoff regime. A short overview of the advantages of such a sensor is discussed here, along with the engineering goals and field testing of a recently built prototype.

Improvised explosive devices (IEDs) in the form of suicide bombers, roadside bombs, and vehicle-borne IEDs (VB-IEDs) are a persistent threat to U.S. troops in the current war on terror. However, detecting and preventing the use of these devices has proven difficult, primarily because the materials to build such devices are readily available to the would-be terrorist and are often commonly used for nonexplosive activities. Stopping the transport of bomb-building materials or locating IED factories would go a long way in preventing the creation of IEDs, which is preferable to detecting and defending against a single IED threat. Unfortunately, few reliable techniques exist for finding explosives in transit, and expert bomb makers cleverly conceal their IED laboratories. 

Detecting bomb-making materials as cargo or finding IED labs is difficult because such materials may present themselves in inconspicuous forms or may be present only in trace amounts away from the actual source. Unlike a carry-on bag at the airport, where explosives can be detected through the use of X-ray and related imaging technologies, penetrating radiation technologies are much more difficult to deploy in a combat theater, and they require potentially unsafe levels of radiation that can penetrate a variety of materials. Moreover, such technology may miss small amounts of trace material in fingerprints or spillage that would be much easier to detect if a bomb maker or transporter of explosives had less than impeccable hygiene. Trace-explosive detection technologies would be the tool of choice in such a scenario.  

At present, the most commonly used techniques for trace explosives detection are canines and variants of ion mobility spectrometry (IMS) and mass spectrometry (MS). Although these methods are very sensitive to trace explosives, they place the instrument and operator in very close proximity to the device. Additionally, canines require lengthy training, need rest periods, and can be distracted. In the cases of IMS and MS, the sample must be introduced into the instrument with a swipe or air sampling, which creates well-known sampling problems, such as cross-contamination, detector saturation, or excessive analysis times.  

Because of the desire for standoff capabilities and real-time detection, laser-based techniques have been proposed as alternatives to the aforementioned methods. Some laser-based methods can analyze explosives from a safe standoff distance of several meters. Moreover, laser-based methods can offer sensitivity and specificity similar to IMS instruments under certain circumstances without the sampling issues previously mentioned.  

Of the possible laser-standoff methods available, laser-induced breakdown spectroscopy (LIBS) and Raman spectroscopy have emerged as two of the top contenders for trace-quantity explosive detection (1–3). However, each technique has its own strengths and weaknesses in the areas of sensitivity, selectivity, and standoff capabilities. For example, LIBS offers high sensitivity to molecule types, but lacks specificity. The abilities of Raman spectroscopy fall at the other extreme. 

Some of the pitfalls of LIBS and Raman spectroscopy can be remedied by enhancing them individually and by combining them into a single instrument. For example, LIBS signals can be enhanced significantly in a dual laser pulse technique called Townsend effect plasma spectroscopy (TEPS) (4) to extend the standoff distance and sensitivity. When combined with multiwavelength Raman for increased selectivity, a single sensor sharing much of the same hardware offers a higher probability of finding explosive residues than TEPS or single-wavelength Raman alone (5). Further advantages of the multitechnique approach and its use in a current system are discussed here. 

Alakai (Largo, Florida) has designed a prototype sensor platform called the Checkpoint Explosives Detection System (CPEDS) using Raman and TEPS hardware for explosives detection at primarily military checkpoints. The system operates from standoff distances for the analysis of trace residues on surfaces and substances related to the manufacture of IEDs. All components of the system are stored inside a customized military S-250 shelter for protection against dust and environmental variation. Because of this compartmentalization, it can be loaded on the back of a high-mobility multipurpose wheeled vehicle (HMMWV) for transport to investigations of potential IED-building laboratories (Figure 1).  

Figure 1: The Alakai CPEDS system on the back of a military HMMWV. 

Figure 2 shows a simplified laboratory setup for a multisensor system like that in CPEDS, but in actual practice, an automated system of optics and actuators is needed for practical operation of any laser spectroscopy system in a standoff regime. For example, CPEDS has an actuated turret that contains a window that serves as the exit and entrance port of light from the system. Entering light is passed to a computer-controlled spectroscopic system. The exiting light (that is, the laser light) is focused with electronically actuated optics and is aligned to cameras inside the system and on the turret. The output of these cameras is shown on a computer that can control the spectrometer and data analysis settings for the currently selected technique from several meters away by means of computer networking and custom software (Figure 3).  

Combining multiple sensors into one instrument increases the probability of detecting explosive residues and enables the detection of a greater number of explosives.

Special Issues-04-01-2011

Advances in Spectroscopy for Detection and Identification of Potential Bioterror Agents

Monitoring of Biological Matrices by GC–MS-MS for Chemical Warfare Nerve Agent Detection

Detecting Explosives by Portable Raman Analyzers: A Comparison of 785-, 976-, 1064-, and 1550-nm (Retina-Safe) Laser Excitation

Detection of Chemicals with Standoff Raman Spectroscopy

Explosives Sensing Using Multiple Optical Techniques in a Standoff Regime with a Common Platform

Mid-Infrared Vibrational Spectroscopy Standoff Detection of Highly Energetic Materials: New Developments

Vol 26 No s4 Spectroscopy April 2011 Defense and Homeland Security Supplement PDF