Ashish Tripathi

Critical role of adsorption equilibria on the determination of surface-enhanced Raman enhancement.

Surface-enhanced Raman spectroscopy (SERS) is a useful technique for probing analyte-noble metal interactions and determining thermodynamic properties such as their surface reaction equilibrium constants and binding energies. In this study, we measure the binding equilibrium constants and Gibbs free energy of binding for a series of nitrogen-containing aromatic molecules adsorbed on Klarite substrates. A dual Langmuir dependence of the SERS intensity on concentration was observed for the six species studied, indicating the presence of at least two different binding energies. We relate the measured binding energies to the previously described SERS enhancement value (SEV) and show that the SEV is proportional to the traditional SERS enhancement factor G, with a constant of proportionality that is critically dependent on the adsorption equilibrium constant determined from the dual Langmuir isotherm. We believe the approach described is generally applicable to many SERS substrates, both as a prescriptive approach to determining their relative performance and as a probe of the substrates affinity for a target adsorbate.

Trace explosive detection in fingerprints with Raman chemical imaging

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.

Classification of select category A and B bacteria by Fourier transform infrared spectroscopy

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.

Standard method for characterizing SERS substrates

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.

Proximal and point detection of contaminated surfaces using Raman spectroscopy

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.

Analytical SERS: general discussion

George Schatz opened a general discussion of the paper by Zhong-Qun Tian: The dependence of Raman intensities with the angle of incidence and angle of scattering is an important issue. This was descirbed for flat surfaces long ago (before SERS) by Greenler and Schlager. How do your results differ?

Biomolecule Raman spectral temporal flux from resting bacillus spores in deionized water matrix

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.

Fate Study of Water Borne Gram Positive Vegetative Bacterial Cells with Raman Microscopy

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.

Water Matrix and Age Effects on Microorganism Raman Microspectroscopy

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.

Bacterial mixture analysis with Raman chemical imaging microspectroscopy

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 Pearsons 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.