Steven D. Christesen

Bacillus Spore Classification via Surface-Enhanced Raman Spectroscopy and Principal Component Analysis

Surface-enhanced Raman spectroscopy (SERS) can provide rapid fingerprinting of biomaterial in a nondestructive manner. The adsorption of colloidal silver to biological material suppresses native biofluorescence while providing electromagnetic surface enhancement of the normal Raman signal. This work validates the applicability of qualitative SER spectroscopy for analysis of bacterial species by utilizing principal component analysis (PCA) to show discrimination of biological threat simulants, based upon multivariate statistical confidence limits bounding known data clusters. Gram-positive Bacillus spores (Bacillus atrophaeus, Bacillus anthracis, and Bacillus thuringiensis) are investigated along with the Gram-negative bacterium Pantoea agglomerans.

Semi-Automated Detection of Trace Explosives in Fingerprints on Strongly Interfering Surfaces with Raman Chemical Imaging

We have previously demonstrated the use of wide-field Raman chemical imaging (RCI) to detect and identify the presence of trace explosives in contaminated fingerprints. In this current work we demonstrate the detection of trace explosives in contaminated fingerprints on strongly Raman scattering surfaces such as plastics and painted metals using an automated background subtraction routine. We demonstrate the use of partial least squares subtraction to minimize the interfering surface spectral signatures, allowing the detection and identification of explosive materials in the corrected Raman images. The resulting analyses are then visually superimposed on the corresponding bright field images to physically locate traces of explosives. Additionally, we attempt to address the question of whether a complete RCI of a fingerprint is required for trace explosive detection or whether a simple non-imaging Raman spectrum is sufficient. This investigation further demonstrates the ability to nondestructively identify explosives on fingerprints present on commonly found surfaces such that the fingerprint remains intact for further biometric analysis.

Raman Chemical Imaging of Explosive-Contaminated Fingerprints

Raman chemical imaging (RCI) has been used to detect and identify explosives in contaminated fingerprints. Bright-field imaging is used to identify regions of interest within a fingerprint, which can then be examined to determine their chemical composition using RCI and fluorescence imaging. Results are presented where explosives in contaminated fingerprints are identified and their spatial distributions are obtained. Identification of explosives is obtained using Pearsons cosine cross-correlation technique using the characteristic region (500–1850 cm−1) of the spectrum. This study shows the ability to identify explosives nondestructively so that the fingerprint remains intact for further biometric analysis. Prospects for forensic examination of contaminated fingerprints are discussed.

Role of the micro- and nanostructure in the performance of surface-enhanced Raman scattering substrates assembled from gold nanoparticles.

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 microfluidic 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 a drastic plunge in substrate performance. The peak intensity does not strongly correlate to the thickness of the nanoparticle films. The results make possible the efficient controlled fabrication of stable, reproducible, and highly active substrates for SERS based chemical sensors with continuous sampling.

Raman Cross Sections of Chemical Agents and Simulants

The differential Raman scattering cross sections of the chemical agents GA, GB, HD, and VX, and the chemical agent simulants DMMP and DIMP have been measured with visible and UV wavelength excitation. These compounds proved to be weak Raman scatterers with cross sections for the strongest lines on the order of 6 to 10 × 10−30 cm2/sr/molecule with 514.5-nm excitation. Excitation at 363.8 nm yielded only a slight preresonance enhancement of the Raman cross sections. Because of the small cross sections and the lack of enhancement, remote Raman detection of chemical agents appears doubtful.

On-Line Spectroscopic Characterization of Sodium Cyanide with Nanostructured Gold Surface-Enhanced Raman Spectroscopy Substrates

To implement surface-enhanced Raman spectroscopy as a practical detection method, highly enhancing, stable, and reproducible substrates need to be fabricated in an efficient manner, and their performance in different solution environments should be well characterized. In this work structured porous gold films have been fabricated using colloidal crystals to template gold nanoparticles. These films were integrated into an on-line flow chamber and used to study the effects of pH and other additives on the detection of sodium cyanide. The gold films proved to be highly enhancing and were used to detect cyanide over a wide range of pH values in the concentration range of ∼2 to 200 ppb. The Raman signal intensity could be increased by lowering the pH after the adsorption of cyanide, which was likely due to both a change in the ionization state and a conformational change of the bound molecules. The peak intensity could also be enhanced multifold by treating the substrate with silver nitrate. Cyanide could be removed from the substrates using hydrochloric acid, although this also passivated the structures, and the activity could only be restored partially with tannic acid. These results provide a rational method to optimize the online detection of cyanide in water.

Characterization of polymorphic states in energetic samples of 1,3,5-trinitro-1,3,5-triazine (RDX) fabricated using drop-on-demand inkjet technology.

The United States Army and the first responder community are evaluating optical detection systems for the trace detection of hazardous energetic materials. Fielded detection systems must be evaluated with the appropriate material concentrations to accurately identify the residue in theater. Trace levels of energetic materials have been observed in mutable polymorphic phases and, therefore, the systems being evaluated must be able to detect and accurately identify variant sample phases observed in spectral data. In this work, we report on the novel application of drop-on-demand technology for the fabrication of standardized trace 1,3,5-trinitro-1,3,5-triazine (RDX) samples. The drop-on-demand sample fabrication technique is compared both visually and spectrally to the more commonly used drop-and-dry technique. As the drop-on-demand technique allows for the fabrication of trace level hazard materials, concerted efforts focused on characterization of the polymorphic phase changes observed with low concentrations of RDX commonly used in drop-on-demand processing. This information is important when evaluating optical detection technologies using samples prepared with a drop-on-demand inkjet system, as the technology may be “trained” to detect the common bulk α phase of the explosive based on its spectral features but fall short in positively detecting a trace quantity of RDX (β-phase). We report the polymorphic shifts observed between α- and β-phases of this energetic material and discuss the conditions leading to the favoring of one phase over the other.

Bioaerosol Analysis with Raman Chemical Imaging Microspectroscopy

Raman chemical imaging microspectroscopy is evaluated as a technology for waterborne pathogen and bioaerosol detection. Raman imaging produces a three-dimensional data cube consisting of a Raman spectrum at every pixel in a microscope field of view. Binary and ternary mixtures including combinations of polystyrene beads, gram-positive Bacillus anthracis, B. thuringiensis, and B. atrophaeus spores, and B. cereus vegetative cells were investigated by Raman imaging for differentiation and characterization purposes. Bacillus spore aerosol sizes were varied to provide visual proof for corroboration of spectral assignments. Conventional applications of Raman imaging consist of differentiating relatively broad areas of a sample in a microscope field of view. The spectral angle mapping data analysis algorithm was used to compare a library spectrum with experimental spectra from pixels in the microscope field of view. This direct one-to-one matching is straightforward, does not require a training set, is independent of absolute spectral intensity, and only requires univariate statistics. Raman imaging is expanded in its capabilities to differentiate and distinguish between discrete 1-6 microm size bacterial species in single particles, clusters of mixed species, and bioaerosols with interference background particles.

Nonintrusive Analysis of Chemical Agent Identification Sets Using a Portable Fiber-Optic Raman Spectrometer

A portable fiber-optic Raman system comprising a diode laser, echelle spectrograph, charge-coupled device (CCD) detector, and filtered fiber-optic probe has been 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 CAIS chemicals, the determination of detection limits for mustard gas and lewisite in chloroform, and the analysis of CAIS items in the field.

Recent Advances and Remaining Challenges for the Spectroscopic Detection of Explosive Threats

In 2010, the U.S. Army initiated a program through the Edgewood Chemical Biological Center to identify viable spectroscopic signatures of explosives and initiate environmental persistence, fate, and transport studies for trace residues. These studies were ultimately designed to integrate these signatures into algorithms and experimentally evaluate sensor performance for explosives and precursor materials in existing chemical point and standoff detection systems. Accurate and validated optical cross sections and signatures are critical in benchmarking spectroscopic-based sensors. This program has provided important information for the scientists and engineers currently developing trace-detection solutions to the homemade explosive problem. With this information, the sensitivity of spectroscopic methods for explosives detection can now be quantitatively evaluated before the sensor is deployed and tested.