KEYWORDS: Raman spectroscopy, Explosives, Luminescence, Signal to noise ratio, Chemical analysis, Sensors, Prototyping, Near infrared, Optical engineering, Hazard analysis
Handheld Raman systems have become powerful analytical tools for the detection and identification of hazardous chemical materials that are now commonly used by both the civilian and military communities. Due to the availability of compact lasers and sensitive detectors, systems typically operate at 785 nm. However, the Raman return at this wavelength can still be obscured by fluorescent impurities in the targeted materials or their matrices. To potentially mitigate this shortcoming, a prototype dual-wavelength Raman incorporating both 785- and 1064-nm excitations was developed and assessed at the Edgewood Chemical Biological Center. The results of that evaluation are discussed here.
We present the methodology and results of a standard assessment protocol to evaluate disparate SERS substrates that
were developed for the Defense Advanced Research Programs Agency (DARPA) SERS Science and Technology
Fundamentals Program. The results presented are a snapshot of a collaborative effort between the US Army Edgewood
Chemical Biological Center, and the US Army Research Laboratory-Aldelphi Laboratory Center to develop a
quantitative analytical method with spectroscopic figures of merit to unambiguously compare the sensitivity and
reproducibility of various SERS substrates submitted by the program participants. We present the design of a common
assessment protocol and the definition of a SERS enhancement value (SEV) in order to effectively compare SERS active
surfaces.
We are actively investigating the use of Raman spectroscopy for proximal standoff detection
of chemicals and explosive materials on surfaces. These studies include Raman Chemical Imaging of
contaminated fingerprints for forensic attribution and the assessments of commercial handheld or
portable Raman instruments operating with near-infrared (IR) as well as ultraviolet (UV) laser
excitation specifically developed for on-the-move reconnaissance of chemical contamination. As
part of these efforts, we have measured the Raman cross sections of chemical agents, toxic industrial
chemicals, and explosives from the UV to NIR. We have also measured and modeled the effect
interrogation angle has on the Raman return from droplets on man-made surfaces. Realistic droplet
distributions have been modeled and tested against variations in surface scan patterns and laser spot
size for determining the optimum scan characteristics for detection of relevant surface
contamination.
Ultraviolet resonance Raman spectroscopy (UVRRS) has been used to examine a variety of different isomers of
nitroaromatic molecules. Due to the large cross section enhancements possible, UVRRS has the potential to be a
sensitive means for detecting trace quantities of explosives at standoff distances. Since it probes both the electronic and
vibrational states of the molecules, it can also be a selective means for differentiating between similar molecules.
Resonance Raman spectra will be discussed, along with the different trends that are observed, for the different positional
isomers of dinitrobenzene. In addition, spectra for the common explosive 2,4,6-trinitrotoluene will be presented.
Fielded surface detection systems rely on contact with either the liquid contamination itself or the associated chemical
vapor above the contaminated surface and do not provide a standoff or remote detection capability. Conversely, standoff
chemical vapor sensing techniques have not shown efficacy in detecting those contaminants as liquids or solids on
surfaces. There are a number of optical or spectroscopic techniques that could be applied to this problem of standoff
chemical detection on surfaces. The three techniques that have received the most interest and development are laser
induced breakdown spectroscopy (LIBS), fluorescence, and Raman spectroscopy. Details will be presented on the
development of these techniques and their applicability to detecting CBRNE contamination on surfaces.
Raman spectroscopy is a very powerful technique for molecular identification, and small Raman instruments have been
used successfully to identify toxic substances. The sensitivity of the technique, however, can be limited by fluorescence
interference arising from the analyte itself or sample impurities. In the case of surface detection, the Raman signature
and/or fluorescence from the surface can also interfere with identification of the target chemical. We take advantage of
the polarization characteristics of the Raman scattering to reduce the broadband fluorescence background and surface
Raman features. Using a custom fiber optic probe with excitation at 785 nm, we have demonstrated real-time
polarization analysis. The spectrum obtained by ratioing the parallel and perpendicular polarization components of the
Raman scattering, reduces the surface signature and has a better spectral correlation to the target analyte.
We present an initial bacterial fate study of Gram positive vegetative cells suspended in water and stored at
ambient room temperature via Raman spectroscopy monitoring. Two types of cells were considered for this
study: vegetative cells of Bacillus cereus, Bacillus thuringiensis which contain the polyhydroxybutyric acid
(PHBA) as an energy storage compound and Bacillus subtlilis cells which do not. The cells were cultured
specifically for this project. Immediately following the culturing phase, the bacteria were extracted, cleaned
and at the onset of the study were suspended in de-ionized water and stored at room temperature. Aliquots of
suspensions were deposited onto aluminum slides at different times and allowed to dry for Raman analysis.
Spectra from multiple regions of each dried spot and each deposit time were acquired along with the bright-field
and fluorescence images. Results were examined to investigate the effect of suspension time on the
spectral signatures as well as the fate behavior of the three types of cells investigated. The cells were
monitored daily for over a 14 period during which time the onset of starvation induced sporulation was
observed.
A novel microfluidic/SERS platform has been developed for real time sensing of 2,4-DNT. The fundamental
research is being conducted at UCSB, commercialized by SpectraFluidics, and validated at ECBC. The system
leverages phenomena at multiple length scales, ranging from tens of micrometers to several nanometers. The key
enabling technology is a newly developed invention termed Free-Surface Fluidics (FSF), where one or more fluidic
surfaces are confined by surface tension forces, and exposed to the surrounding atmosphere. The free-surface fluidic
architecture is combined with surface-enhanced Raman spectroscopy (SERS) for detection of 2,4-DNT. Once 2,4-DNT
analyte molecules are absorbed into the flow, they can interact with gold or silver colloidal particles. This architecture
allows for analysis and deterministic control of SERS 'hot spot' aggregation, which can increase Raman scattering signal
strength by up to 10 orders in magnitude. We have successfully measured DNT vapor at concentrations as low as ~1
ppb. This sensitivity value is confirmed by orthogonal measurements using GC-mass spectroscopy at ECBC.
Wide-field Raman chemical imaging (RCI) has been used to detect and identify the presence of trace
explosives in contaminated fingerprints. A background subtraction routine was developed to minimize the
Raman spectral features produced by surfaces on which the fingerprint was examined. The Raman image was
analyzed with a spectral angle mapping routine to detect and identify the explosives. This study shows the
potential capability to identify explosives non-destructively so that the fingerprint remains intact for further
biometric analysis.
Fielded surface detection systems rely on contact with either the liquid contamination itself or the associated chemical
vapor above the contaminated surface and do not provide a standoff or remote detection capability. Conversely, standoff
chemical vapor sensing techniques have not shown efficacy in detecting those contaminants as liquids or solids on
surfaces. There are a number of optical or spectroscopic techniques that could be applied to this problem of standoff
chemical detection on surfaces. The three techniques that have received the most interest and development are laser
induced breakdown spectroscopy (LIBS), fluorescence, and Raman spectroscopy. Details will be presented on the
development of these techniques and their applicability to detecting CBRNE contamination on surfaces.
Raman chemical imaging microspectroscopy (RCIM) is being evaluated as a technology for waterborne
pathogen detection. Binary and ternary mixtures including combinations of polystyrene beads, Grampositive
Bacillus anthracis and B. atrophaeus spores, B. cereus vegetative cells, and Gram-negative E. coli
cells were investigated by RCIM for differentiation and characterization purposes. We have demonstrated
the ability of RCIM, in combination with Pearson's cross correlation and multivariate principal components
analysis data reduction techniques, to differentiate these components in the same field of view (FOV).
Conventional applications of RCIM consist of differentiating relatively broad areas in a FOV. Here, RCIM
is expanded in its capabilities to differentiate and distinguish between different micron size species in
single particles and clusters of mixed species.
UV Raman spectroscopy is being applied to the detection of natural and man-made surfaces contaminated with chemical agents. In support of these efforts, we have measured the UV Raman signatures of chemical agents and their simulants. In addition, we have measured both the UV Raman and UV absorption cross sections of these compounds for determining their relative limits of detection. The UV Raman measurements were made using a doubled Argon ion laser operating at 248 nm. Spectra were collected on an echelle spectrograph equipped with a CCD array detector. Based on the data collected, we also discuss the suitability of currently accepted agent simulants for testing UV Raman detection instruments.
Raman and surface-enhanced Raman spectroscopy (SERS) studies of bacteria have reported a wide range of
vibrational mode assignments associated with biological material. We present Raman and SER spectra of the amino
acids phenylalanine, tyrosine, tryptophan, glutamine, cysteine, alanine, proline, methionine, asparagine, threonine,
valine, glycine, serine, leucine, isoleucine, aspartic acid and glutamic acid and the nucleic acid bases adenosine,
guanosine, thymidine, and uridine to better characterize biological vibrational mode assignments for bacterial target
identification. We also report spectra of the bacteria Bacillus globigii, Pantoea agglomerans, and Yersinia rhodei
along with band assignments determined from the reference spectra obtained.
Surface-enhanced Raman scattering (SERS) provides rapid fingerprinting of biomaterial in a non-destructive manner. The problem of tissue fluorescence, which can overwhelm a normal Raman signal from biological samples, is largely overcome by treatment of biomaterials with colloidal silver. This work presents a study into the applicability of qualitative SER spectroscopy with principal component analysis (PCA) for the discrimination of four biological threat simulants; Bacillus globigii, Pantoea agglomerans, Brucella noetomae, and Yersinia rohdei. We also demonstrate differentiation of gram-negative and gram-positive species and as well as spores and vegetative cells of Bacillus globigii.
Raman spectroscopy is just one of the diverse set of detection techniques which, under the CB Rapid Agent Aerosol Detection (RAAD) program, are being evaluated for their ability to detect and identify biological materials. In order to compare and contrast different techniques, a Common Sample Set composed of threat simulants, interferents and growth media was provided to all RAAD participants. The samples were investigated using both normal Raman and surface-enhanced Raman spectroscopy. This paper focuses on near-infrared Raman data from the Common Sample Set bacterial simulants. Results are also given from a principal component analysis performed on these samples. These measurements provide and initial assessment of the detection and discrimination capability of Raman spectroscopy as applied to biological materials. Despite the challenges facing this detection method, Raman spectroscopy is emerging as a rapid and information-rich method of investigating biological threats.
This work presents the development of new methodologies centering on surfaces with immunologically induced affinities for biomaterials in aqueous systems. The immunologically active surfaces concentrate the biomaterials at the interface and therefore eliminate the need for preconcentration steps. This results in a highly sensitive and rapid immunoassay technique. The very strong localized of surface enhanced Raman scattering (SERS) that occurs at noble metal surfaces is combined with the unparalleled selectivity of immunoassays. Localization of the SERS signal eliminates the problem of washing and allows assays to be performed without treatment steps associated with removing excess agents. Previous work with small illicit drug molecules and large microorganisms clearly demonstrates trace detection of species in aqueous environments is possible. This paper discusses further work to detect Bacillus globigii by couping surface enhanced Raman scattering with immunoassays (SERIA) using citrate reduced silver nanoparticles. The spores of B. globigii are used to simulate the behavior of another bacterium that forms spores-the potential biological warfare agent, Bacillus anthracis, the causative agent of anthrax.
Highly active and stable substrates for surface-enhanced Raman scattering (SERS) can be fabricated by using colloidal crystals to template gold nanoparticles into structured porous films. The structure-dependent performance of these SERS substrates was systematically characterized with cyanide in continuous flow millifluidic chambers. A matrix of experiments was designed to isolate the SERS contributions arising from nano- and microscale porosity, long range ordering of the micropores, and the thickness of the nanoparticle layer. The SERS results were compared to the substrate structure observed by scanning electron microscopy (SEM) and optical microscopy to correlate substrate structure to SERS performance. The Raman peak intensity was consistently highest for nanoporous substrates with three-dimensionally ordered micropores, and decreases if the micropores are not ordered, or not templated. Removing the nanoscale porosity by fusion of the nanoparticles (without removing the large micropores) leads to drastic plunge in substrate performance. The peak intensity does not strongly correlate to the thickness of the nanoparticle films. Receiver operating characteristic (ROC) curve analysis for cyanide in water revealed a limit of detection (LOD) of ca. 150 ppb based on a 5% probability of false alarm. The results make possible the efficient controlled fabrication of stable, reproducible and highly active substrates for SERS-based chemical sensors with continuous sampling.
Resonance Raman spectroscopy is an enhanced Raman technique that can be used to selectively identify a particular analyte in complex matrices. Resonance Raman requires the excitation laser to overlap with an absorption band of the analyte of interest. Since analytes have diverse absorption spectra, dilute concentrations may be detected when resonantly enhanced. A significant portion of interesting molecules absorb only in the UV; unfortunately current UV Raman instrumentation for scientifically desirable spectral resolution is large and costly. In the area of Homeland Defense, explosives, nerve agents, amino acid residues (for toxin analysis) and nucleic acids (for DNA detection and identification of bacteria) are all enhanced using UV laser sources. EIC Laboratories has developed a more user-friendly UVRRS spectrograph that is based upon the use of an echelle grating. The spectrograph has a footprint of 7" x 11" and is capable of providing 4 cm-1 resolution over a fairly wide spectral range. The spectrograph design and spectra from analytes of particular relevance will be presented.
Brookhaven National Laboratory (BNL), Edgewood Chemical and Biological Center (ECBC) and ITT Industries Advanced Engineering and Sciences Division (AES) have been collaborating on the transitioning and subsequent development of a short-range, non-contact Raman lidar system specifically designed to detect and identify chemical agents on the battlefield. [The instrument, referred to as LISA (Laser Interrogation of Surface Agents), will the subject of an accompanying paper.] As part of this collaboration, BNL has the responsibility for developing a spectral database (library) of surrogates and precursors for use with LISA’s pattern recognition algorithms. In this paper, the authors discuss the phenomenon of UV Raman and resonance-enhanced Raman spectroscopy, the development of an instrument-independent Raman spectral library, and highlight the exploitable characteristics present in the acquired spectral signatures that suggest potential utility in our country’s efforts on Homeland Security.
Surface-enhanced Raman spectroscopy (SERS) is a spectroscopic technique that enables trace detection of analytes of relevance using fieldable equipment. SERS uses the enhanced Raman signals observed when an analyte adsorbs to a roughened metal substrate, generally gold, silver, or copper. Coupled to a microscope, single molecule detection has been demonstrated. With a fieldable instrument, enhancements of 108 compared to unenhanced Raman spectroscopy are expected, allowing trace detection in the field. Proper development of the metal substrate will optimize the sensitivity and selectivity towards the analytes of interest. In this presentation, we will discuss applications under development at EIC Laboratories that are of importance to Homeland Defense. We will review the capabilities of SERS to detect buried explosives, explosives associated with nuclear weaponry and chemicals involved in the nuclear enrichment process. We will discuss the detection of chemical and biological warfare agents in the water supply in research performed under the Joint Service Agent Water Monitor. We will demonstrate the current detection limits, the reproducibility of the signal, and results collected using actual chemical warfare agents, and show how the results can be extended to vapor detection. We will also discuss the current state-of-the art for fieldable instrumentation. The emphasis on portability and speed will be stressed; SERS acquisitions are restricted to 30 s or less.
In the past decade, the Unites States and its allies have been challenged by a different kind of warfare, exemplified by
the terrorist attacks of September 11, 2001. Although suicide bombings are the most often used form of terror, military
personnel must consider a wide range of attack scenarios. Among these is the intentional poisoning of water supplies to
obstruct military operations in Afghanistan and Iraq. To counter such attacks, the military is developing portable
analyzers that can identify and quantify potential chemical agents in water supplies at microgram per liter
concentrations within 10 minutes. To aid this effort we have been investigating the value of a surface-enhanced Raman
spectroscopy based portable analyzer. In particular we have been developing silver-doped sol-gels to generate SER
spectra of chemical agents and their hydrolysis products. Here we present SER spectra of several chemical agents
measured in a generic tap water. Repeat measurements were performed to establish statistical error associated with
SERS obtained using the sol-gel coated vials.
Protection of the drinking water supply from a terrorist attack is of critical importance. Since the water supply is vast, contamination prevention is difficult. Therefore, rapid detection of contaminants, whether a military chemical/biological threat, a hazardous chemical spill, naturally occurring toxins, or bacterial build-up is a priority. The development of rapid environmentally portable and stable monitors that allow continuous monitoring of the water supply is ideal. EIC Laboratories has been developing Surface-Enhanced Raman Spectroscopy (SERS) to detect chemical agents, toxic industrial chemicals (TICs), viruses, cyanotoxins and bacterial agents. SERS is an ideal technique for the Joint Service Agent Water Monitor (JSAWM). SERS uses the enhanced Raman signals observed when an analyte adsorbs to a roughened metal substrate to enable trace detection. Proper development of the metal substrate will optimize the sensitivity and selectivity towards the analytes of interest.
Laser Interrogation of Surface Agents (LISA) is a UV-Raman technique that provides short-range standoff detection and identification of surface-deposited chemical agents. ITT Industries, Advanced
Engineering and Sciences Division, is currently developing and expanding the LISA technology under several programs that span a variety of missions for homeland defense. We will present and discuss some of these applications, while putting in perspective the overall evolution undergone by the technique within the last years. These applications include LISA-Recon (now called the Joint Contaminated Surface Detector--JCSD) which was developed under a cost-sharing arrangement with the U.S. Army Soldier and Biological Chemical Command (SBCCOM) for incorporation on the Army’s future reconnaissance
vehicles, and designed to demonstrate single-shot on-the-move measurements of chemical contaminants at concentration levels below the Army's requirements. In parallel, LISA-Shipboard is being developed to optimize the sensor technique for detection of surface contaminants in the operational environment of a ship. The most recently started activity is LISA-Inspector that is being developed to provide a transportable sensor in a 'cart-like' configuration.
Surface-enhanced Raman spectroscopy (SERS) is a highly sensitive technique for quantifying trace amounts of analyte adsorbed at a roughened metal surface. Many techniques, including electrochemical etching and e-beam lithography, have been used previously to produce roughened metallic surfaces. In this work we demonstrate how novel gold nanostructured films, which are simply fabricated using gold nanoparticles and latex microspheres, can be used as highly sensitive SERS substrates. The gold films are templated by 3D colloidal crystals and display long ranged ordered regions. Since the films are porous on two length scales and, therefore, possess a high surface area, we have investigated their SERS activity using sodium cyanide as a model compound. We have integrated these substrates into a flow chamber and demonstrated the quantitative detection of sodium cyanide form 5 to 500 ppb. Our results also reveal that cyanide detection can be significantly enhanced by lowering the pH after cyanide adsorption, likely indicating a conformational change of the bound cyanide. This study illustrates how novel materials formed by simple wet chemistry techniques can be used in practical devices for the detection of chemical agents, and, more generally, illustrates how material design and spectroscopic evaluation can be rapidly integrated.
The recent distribution of anthrax through the U.S. postal system and the subsequent infection and death of several postal and national media employees, amplifies the need for methods to rapidly detect, identify, and quantify this and other chemical and biological warfare agents. The U.S. military has also identified water supplies as a likely method of warfare agent deployment and is funding the development of a Joint Service Agent Water Monitor. In an effort to aid military personnel and the public at large, we are developing a portable analyzer capable of identifying and quantifying chemical agents rapidly, either 'on-demand' or continuously.
Portable fiber-optic Raman systems are being used to analyze chemical agents and other toxic chemicals in sealed glass containers. These containers include ampoules and bottles that are contents of chemical agent identification sets (CAIS) developed for use in training military personnel in chemical agent identification, safe handling, and decontamination. Real-time nonintrusive analysis of these sets is required so that the items containing chemical agents can be identified for proper disposal. This paper details the laboratory measurement of Raman spectra of chemical agents, Raman scattering cross sections of chemical agents, and the analysis of CAIS items in the field.
A UV fluorescence lidar system for the remote detection of bioaerosols has been built and tested. At the heart of the UV- LIDAR Fluorosensor system are a 200 mJ quadrupled Nd:YAG laser at 266 nm and a 16-inch Cassagrain telescope. Operating on three data collection channels, the UV lidar is capable of real time monitoring of 266 nm elastic backscatter, the total fluorescence between 300 and 400 nm, and the dispersed fluorescence spectrum (using a small spectrograph and gated intensified CCD array). Our goal in this effort was to assess the capabilities of biofluorescence for quantitative detection and discrimination of bioaerosols. To this end, the UV-LIDAR Fluorosensor system was tested against the aerosolized bacterial spore Bacillus subtilus var. niger sp. globiggi (BG) and several likely interferences at several ranges from approximately 600 to 3000 m. Our tests with BG indicate a detection limit of approximately 500 mg/cubic meter at a range of 3000 m.
Until quite recently the ability to detect and discriminate aerosolized micro-organisms at long range using the laser induced fluorescence (LIF) technique has met with limited success. The lasers which met our logistic requirements had insufficient energies to propagate through the troposphere and excite a target organism. The detectors, though sensitive enough, did not allow us to see a spectral distribution of the fluorescence return. Advances in laser and detector technology has now brought us higher energy, solid state lasers, and very sensitive array detectors. Using this new technology we built and tested an ultraviolet LIDAR against various interferents and a micro-organic contaminant. In this paper we describe the system and method used to detect and discriminate an aerosolized micro-organism at ranges up to 3 kilometers, and the results of this effort.
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