Transitioning a technical method from the laboratory bench to the field is a challenge. Initially, the method needs to fill a technical gap to a degree that a warfighter or first responder would find additional hardware and training worth the logistical burden. Second, the method should be robust to minor deviations and interferents. Finally, the resultant end point must be easily read, understood, and provide actionable information to the user. Accomplishing all these steps is key to demonstrating the value of scientific research to the warfighter and delivering a valuable tool. Recent efforts have been focused on developing methods for easy and robust trace analyte collection and portable sample identification. The analytes of interest include explosives, pharmaceutical based agents, and drugs of abuse. The collection method involves paper modified with pressure-sensitive adhesives, i.e. yellow sticky notes, to sample various types of solid, porous, and environmental surfaces. Threat identification is performed directly from the collection substrates by mass spectrometric instrumentation with tandem capabilities to identify TNT, RDX, and HMX. The surface limits of detection (LODs) of the method ranged from sub to low microgram range. An analysis mode was created that would display a green light/red light if a sample was negative/positive, respectively, for a threat. This provides an easy-to-read, actionable result while saving the analytical spectra for future review. Finally, this methodology was combined with portable Raman analysis to provide both primary and confirmatory identification of fentanyl in simulated samples and TNT in samples both collected and analyzed in an austere location.
Waveguide-enhanced Raman spectroscopy (WERS) using nanophotonic waveguides has been used to demonstrate the detection of vapor-phase chemicals and liquid-phase biomolecules in water. The technique benefits from the fabrication processes and tolerances of CMOS foundries, but successful foundry-based WERS photonic integrated circuits (PICs) have only been demonstrated using excitation wavelengths of 1064 nm and 785 nm. Foundry-based PICS are beginning to operate with low loss at visible wavelengths, and WERS is uniquely poised to take advantage of this capability. Raman scattering cross-sections scale as λ−4, so a visible WERS platform could enable increased sensitivity, decreased exposure times, and/or decreased laser powers. However, increased fluorescence, increased waveguide loss, and decreased feature sizes make WERS in the visible challenging. Here, we demonstrate WERS using 300-mm foundry-based fabrication (AIM Photonics) with 633 nm and 785 nm laser excitation. We also show the successful operation and integration of other required components for a compact WERS system operating in the visible, including edge-couplers and lattice filters.
Under the U.S. Army Forensics Advanced Research Program, the The Portable Chemical Fingerprint Identification System (P-CFIS) is being developed by U.S. Army Combat Capabilities Development Command –Chemical Biological Center (DEVCOM-CBC) enabling trace level (non-visual) detection of solid particulate contamination on surfaces and residual fingerprints. This operationally flexible non-contact, non-destructive methodology is reducing the need to transport potentially contaminated materials and provide a field forward detection capability yielding greater situational awareness of the threat environment. This uniquely developed prototype based on Raman spectroscopy allow the system to scan a one-inch square area of interest, which may be flat or uneven, target any found particles in the field of view, and automatically analyze and report detection events of threat chemicals contained within the spectral database of the system. This presentation will describe the preliminary results from evaluating the performance of the prototype systems, discussing time of analysis, particle size characteristics, analysis of heterogeneous surfaces, and future development of the systems ultimately leading to the development of the next generation of expeditionary systems for military forensic analysis, checkpoint detection, and/or sensitive site exploitation.
Drop-on-demand and inkjet printing technology continues to be a promising method of producing chemical test standards with scalability and flexibility to allow for inexpensive, high-throughput production of samples. This enabling technique provides precise, accurate and highly reproducible test coupons that mimic the hazardous chemicals encountered in various theater scenarios; critical in assessing the performance of existing and future sensors detection capabilities. Under the U.S. Army Forensics Advanced Research Program, the Spectroscopy Branch within the Research and Technology Directorate, DEVCOM CBC, along with internal and external collaborative partners are currently utilizing the Direct Color Systems 1800z flat-bed inkjet printer for deposition of various chemicals on relevant surfaces and GeSiM NP2.1 Nanoplotter for more precise and control droplet deposition to support various optical and non-optical detection objectives. The samples produced under this project are used for the evaluation of trace level energetic materials and illicit drugs of abuse within latent fingerprints, deposition of sorbent polymers onto photonic integrated circuits for vapor detection, point sensors, and more recently exploring enhanced training aids for military working dogs. This work will present results from the characterization of utilized chemical deposition techniques as well as recent experimental results from various assessed detection technologies
Recent work with B. anthracis Delta Sterne spores demonstrated that Raman spectroscopy could be used to discriminate between viable and gamma deactivated spores and provided initial insight into the probable source of discrimination found in the spores. From this previous work, we believe through Raman spectral analyses of viable and deactivated spore samples, significant changes in spectral response can be resolved and ascribed to classes of biomolecules affected by the deactivation processes. We expanded upon this study to include four different Bacillus spores (B. anthracis, B. megaterium, B. thuringiensis, and B. atrophaeus) and probe de-activation techniques to include gamma radiation UV radiation, chemical, and thermal methodologies. We used sequential Raman imaging scanning electron (RISE) microscopy to determine chemical (Raman spectral information) and physical (SEM imaging) variance between viable and deactivated spore samples. Additional use of machine learning algorithms to
Detection of threat materials is an important capability for the military and homeland security to protect soldiers and civilians. Waveguide-enhanced Raman spectroscopy (WERS), a photonic integrated circuit sensing methodology, is being developed for field detection of materials related to chemical warfare agents, explosives, and narcotic threats. Low-fluorescence silicon nitride spiral waveguides with long path lengths are used to obtain high signal levels with nearinfrared excitation (785 nm and 1064 nm). Compact single-mode-fiber-coupled spectrometers with high sensitivity are being utilized for detection of the Raman scattered light. Thermoelectrically cooled charged coupled device (CCD) or InGaAs detectors (-15 °C) provide for low-noise and high-quantum-efficiency spectral measurement. Performance comparable to that obtained with large benchtop spectrometers is observed. The spiral waveguides are coated with functionalized polymer sorbents suitable for concentrating relevant classes of threat materials in the evanescent field of the waveguide. The sorbents are deposited using piezoelectric microdispensers to allow for controlled deposition of thin films without the need for spin-coating. Raman chemical imaging microscopy is used to characterize the uniformity of the sorbent polymers on the waveguides. Library spectral matching can be used in combination with the selectivity of the sorbent materials to provide discrimination of the materials absorbed by the polymer coatings. The ultimate objective is development of a prototype handheld WERS sensor system suitable for defense and security applications in the field. WERS development and spectral measurements will be presented.
The US Army Research, Development and Engineering Command – Chem Bio center is leading an inter-agency working group, to expand chemical inkjet printing techniques, and to fabricate surface standards in a controlled, uniform and quantifiable fashion, for the evaluation of stand-off active and passive optical systems. A CommercialOff-the-Shelf (COTS) standard inkjet printer was redesigned to deposit precise amounts of chemicals and explosive material on defense relevant surfaces, allowing for the generation of calibration test standards. RDECOM-CB is currently utilizing the inkjet techniques to support an Army forensics detection program where inkjet samples are used for detection of trace energetic materials and illicit drugs of abuse within residual latent fingerprints, as well as leading a North Atlantic Treaty Organization (NATO) Task Group (TG) to develop and recommend to NATO a reference standard methodology (or methodologies) for fabricating quantifiable surface standards for the evaluation of stand-off active and passive optical systems. QA/QC were performed on printed materials to determine accuracy and precision. Raman imaging and the Image-J software package was used to calculate particle statistics such as size distribution, average particle size, and fill factor. The software algorithm finds individual particles and calculates their area from a brightfield image montage. An approximate diameter of each particle, and the total fractional area of the surface covered are also calculated. For qualitative analysis Raman Chemical Imaging is performed to confirm the chemical make-up of the deposited samples. For the quantitative analysis, printed samples were analyzed by either Ion Chromatography with Conductivity Detection (IC-CD) for potassium chlorate based explosives analysis or LC-MS/MS for RDX analysis. We will present the results of inkjet samples produced for the Army forensics program as well as NATO benchmark exercise that consisted of printing trace amounts of inkjet explosive samples and performing QA/QC procedures to determine accuracy, precision and mass transport efficiency.
The U.S. Army Research Development Engineering Command Chemical Biological Center (RDECOM C&B) continues to develop technologies for the forensic detection of energetic materials and illicit drugs of abuse due to their recent confluence in counter terrorism operations. One specific technology developed here is the use of Raman Chemical imaging to detect these substances located concomitant with residual latent fingerprints. This study demonstrates the ability to identify threat materials non-destructively so that the fingerprint remains intact for further biometric analysis. Utilizing Raman spectroscopy, the Generation I Chemical Fingerprint Identification System (CFIS) semi-autonomously locates and identifies particles of interest found on the friction ridge of a given recovered fingerprint with minimal input from the operator. This work presents results from a collaborative effort between the U.S. Defense Forensic Science Center (DFSC) and RDECOM C&B in which two prototype CFIS systems were assessed with a variety of samples and examines additional practical considerations leading toward the development of the next generation of expeditionary systems for military forensic analysis.
Matrix assisted laser desorption ionization (MALDI) is a powerful technique that improved the mass spectrometry (MS) characterization of biological molecules. However this technique requires the mixing of matrix compound with the analyte of interest. The matrix compound used in MALDI process is not universal and usually depends heavily on the nature of analyte of interest being analyzed. As such there are many matrices that are used and without knowing the nature of your analyte it will be hard to predict which matrix is optimal for the most effective MALDI-MS analysis. Moreover, a high energy laser exposure is needed to initiate the ionization process through a charge transfer process between the matrix and analyte molecules. Recent advancement in the metalorganic framework (MOF) field introduced desirable surfaces that can be modified for various applications. Such MOFs can be synthesized with porous solid, and could have regular or predicted geometry. This project is introducing a novel idea of utilizing a modified MALDI substrate with MOF that can provide charge transfer between immobilized functionalized groups and analyte molecules that mimic the solvation process when a solution matrix is used. Begin the abstract two lines below author names and addresses.
Raman microspectroscopy and principal component analysis are used to decipher unique biomolecular information by
monitoring the effect of residence time of Bacillus spores suspended in deionized water. Suspensions of viable spores of
Bacillus anthracis Sterne (BA), Bacillus atrophaeus (BG), and Bacillus thuringiensis were prepared and spectrally
monitored from initial deposition (time zero) and intermittently for seven days. Questions addressed include if spectral
variations are significant with bacterial species and residence time under non-germination conditions, is the
discrimination capability affected, and are there markers indicating pre-germination activity. Clear spectral distinction
for the spore suspensions was observed with respect to residence time, however, when the residence time data were
combined, discrimination analyses showed significant overlap between the BA and BG spores. Temporal spectral
analyses at select wavenumbers suggest an increase in pre-germination activity from the freshly suspended to one day
suspensions.
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.
Raman microspectroscopy is used to probe the age and milieu parameters for suspensions of bacteria for their
detection in water backgrounds. No studies have been reported on the fate of Raman signatures over time for
biologicals stored in water matrices. A FALCON II Raman Chemical Imaging System (ChemImage, Pittsburgh, PA)
and 532 nm laser excitation source acquired the Raman spectra. MATLAB principal components (PC) analysis
software was employed for data reduction. Suspensions of Bacillus atrophaeus, Bacillus thuringiensis, and three
strains of E. coli (EC) were prepared in distilled and recipe tap water. Aliquots at 5 min, 5 hr, and 1, 2, and 7 days at
25 C were dried on microscope slides in replicate. Adequate spectral differences were observed for all three
organism species. Microscope analysis showed that freshly suspended Bacillus spores and EC vegetative cells, in
both water matrices, remained as spores after seven days. Agar plate growth procedures showed that the bacteria
were still viable even after seven days resting in both water matrices. All three bacterial species were separated
based on PC analysis; however, the three EC strains coalesced. The water matrix parameter was inconsistent in its
ability to separate the Raman spectra in PC plots of the five bacteria. Within each group, the time parameter poorly
separated the bacterial resting suspensions as the aging proceeded. A Mahalanobis linkage distance analysis
(dendrogram) for all three species and strains in both water matrices confirmed a random order for all five
suspension times.
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.
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.
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.
Relatively few reports have investigated the determination and classification of pathogens such as the
National Institute of Allergy and Infectious Diseases (NIAID) Category A Bacillus anthracis spores and
cells (BA), Yersinia species, Francisella tularensis (FT), and Category B Brucella species from FTIR
spectra. We investigated the classification ability of the Fourier transform infrared (FTIR) spectra of viable
pathogenic and non-pathogenic NIAID Category A and B bacteria. The impact of different growth media,
growth time and temperature, rolling circle filter of the data, and wavelength range were investigated for
their microorganism differentiation. Various 2-D PC plots provided differential degrees of separation with
respect to the four viable, bacterial genera including the BA sub-categories of pathogenic spores, vegetative
cells, and nonpathogenic vegetative cells. FT spectra were separated from that of the three other genera.
The BA pathogenic spore strains 1029, LA1, and Ames were clearly differentiated from the rest of the
dataset. Yersinia species were distinctly separated from the remaining dataset and could also be classified
by growth media. This work provided evidence that FTIR spectroscopy can separate the four major
pathogenic bacterial genera of NIAID Category A and B biological threat agents.
Raman spectroscopy is being evaluated as a candidate technology for waterborne pathogen detection and
the fidelity of the Raman spectra of microorganisms with respect to their differentiation at the single cell
level are investigated. Individual entities are investigated in the microscope field of view (FOV) by Raman
chemical imaging microscopy (RCIM). The size of a substance was not found to cause spectral confusion
when collating individual entities in the FOV by multivariate principal components (PCA) and RCIM
methods. Polystyrene (PS) beads in 1-3 micron sizes were collectively grouped together by PCA. Distilled
and recipe tap water matrices produced the proper identification of the PS beads throughout the FOV, and
all PS beads in a FOV were grouped together by PCA. A mixture of Gram-positive Bacillus atrophaeus
spores and Gram-negative E. coli cells were differentiated and distinguished by RCIM.
Raman spectroscopy has been evaluated as a candidate technology for waterborne pathogen detection. Parameters have
been investigated that influence the fidelity of Raman spectra of microorganisms and protein biological substances
including bacterial species and strains, susceptibility to laser induced photodamage, composition of water matrix, and
organism aging in water. An important operating parameter is the laser induced photodamage threshold of a variety of
biological materials. The laser induced photodamage may be minimized by operating a 532 nm continuous wave laser
excitation at laser power densities below 2300 W/cm2 for Gram-positive Bacillus atrophaeus (BG) vegetative cells, 2800
W/cm2 for BG spores, and 3500 W/cm2 for Gram-negative E. coli organisms. Multivariate principal components
analysis was able to discriminate six Gram-positive and Gram-negative organisms as well as five proteins between 5K
and 65K mass units. B. thuringiensis, B. cereus, BG spore and vegetative preparations, and E. coli showed minimal
aging effects when suspended in distilled and tap water. In general, Raman microspectroscopy of biological substances
exhibited minimal spectral variability due to the age of a resting suspension, water matrix, and bacterial strain. The
observed signature variability did not prevent the differentiation and characterization of bacterial genus and species and
protein substances using Raman spectroscopy.
Improvements were made to a pyrolysis-gas chromatography-ion mobility spectrometry stand-alone biodetector to provide more pyrolyzate compound information to the IMS detector module. Air carrier gas flowing continuously through the pyrolysis tube, the rate of air flow, and pyrolysis rate were found to improve the relative quality and quantity of pyrolyzate compounds detected by the IMS detector compare to earlier work. These improvements allowed a greater degree of confidence in the correlation of biological aerosols obtain in outdoor testing scenarios to a standard GC-IMS biological aerosol dataset. The airflow improvement allowed more biomarker compounds to be observed in the GC-IMS data domain for aerosols of Gram-negative Erwinia herbicola (EH) and ovalbumin protein as compared to previous studies. Minimal differences were observed for Gram-positive spores of Bacillus subtilis var. globigii (BG) from that of earlier work. Prior outdoor aerosol challenges dealt with the detection of one organism, either EH or BG. Biological aerosols were disseminated in a Western Canadian prairie and the Py-GC-IMS was tested for its ability to detect the biological aerosols. The current series of outdoor trials consisted of three different biological aerosol challenges. Forty-two trials were conducted and a simple area calculation of the GC-IMS data domain biomarker peaks correlated with the correct bioaerosol challenge in 30 trials. In another 7 trials, the status of an aerosol was determined to be biological in origin. Two additional trials had no discernible, unambiguous GC-IMS biological response, because they were black water sprays. Reproducible limits of detection were at a concentration of less than 0.5 bacterial analyte-containing particles per liter of air (ACPLA). In order to realize this low concentration, an aerosol concentrator was used to concentrate 2000 liters of air in 2.2 minutes. Previous outdoor aerosol trials have shown the Py-GC-IMS device to be a credible detector with response to determining the presence of a biological aerosol. The current series of outdoor trials has provided a platform to show that the Py-PC-IMS can provide information more specific than a biological or non-biological analysis to an aerosol when the time of dissemination is unknown to the operator. The Py-GC-IMS is shown to be able to discriminate between aerosols of a Gram-positive spore, a Gram-negative bacterium and a protein.
A commercially available, hand-held chemical vapor detector was modified to detect Gram-positive Bacillus subtilis var. globigii spores (BG) in outdoor field scenarios. An Airborne Vapor Monitor (AVM) ion mobility spectrometry (IMS) vapor detector was interfaced to a biological sample processing and transfer introduction system. The biological sample processing was accomplished by quartz tube pyrolysis (Py), and the resultant vapor was transferred by gas chromatography (GC) to the IMS detector. The Py-GC/IMS system can be described as a hyphenated device where two analytical dimensions, in series, allow the separation and isolation of individual components from the pyrolytic decomposition of biological analytes. Gram positive spores such as BG contain 5 - 15% by weight of dipicolinic acid (DPA), and picolinic acid is a pyrolysis product of DPA. Picolinic acid has a high proton affinity, and it is detected in a sensitive fashion by the atmospheric pressure-based IMS device. Picolinic acid occupies a unique region in the GC/IMS data domain with respect to other bacterial pyrolysis products. A 1000 to 1, air-to-air, aerosol concentrator was interfaced to the Py-GC/IMS instrument, and the system was placed in an open-air, Western United States desert environment. The system was tested with BG spore aerosol releases, and the instrument was remotely operated during a trial. A Met-One aerosol particle counter was placed next to the Py-GC/IMS so as to obtain a real-time record of the ambient and bacterial aerosol challenges. The presence/absence of an aerosol event, determined by an aerosol particle counter and a slit sampler-agar plate system, was compared to the presence/absence of a picolinic acid response in a GC/IMS data window at selected times in a trial with respect to a BG challenge. In the 21 BG trials, the Py-GC/IMS instrument experienced two true negatives, no false positives, and the instrument developed a software failure in one trial. The remaining 18 trials were true positive determinations for the presence of BG aerosol, and a limit of detection for the Py-GC/IMS instrument was estimated at approximately 3300 BG spore-containing particles.
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