Appendix G           Characterization of chemical warfare G-agent hydrolysis products                      by surface-enha...
In our studies, we have employed metal-doped sol-gels to promote the SERS effect. The porous silica network of thealkoxide...
concentration of 1 mg/mL from solid powders or 0.1% v/v from neat liquids unless noted otherwise. Lowerconcentrations were...
intense 756 cm-1 peak and the unique peak at 1300 cm-1. The former peak clearly corresponds to a nearly pure PCsymmetric s...
substantial shift in peak frequencies occurs for PO3 modes when compared to the Raman spectra. The increasedintensity of t...
contributions. The spectral intensity of this mode and the shift in frequency of the PO3 modes in the SERS spectrasuggest ...
18. Hoffland, L.D., Piffath, R.J., and Bouck, J.B. “Spectral signatures of chemical agents and simulants”, Optical    Engi...
Appendix H Surface-enhanced Raman spectra of VX and its hydrolysis products STUART FARQUHARSON,∗ ALAN GIFT, PAUL MAKSYMIUK...
HO                                                                               O                  H2O                   ...
supported by recent pH dependent SERS studies of MPA, that                      modes are no longer pure PC and can not be...
alkanethiols in the Raman and SERS spectra.25-27 It is also                              PO2S bend, the OPC stretch, and a...
Table II. Tentative vibrational mode assignments for Raman and SERS peaks for VX and its hydrolysis products      MPA     ...
SERS spectra of VX and EA2192 are not that similar. In                     like to thank Dr. Steve Christesen for helpful ...
Appendix I                                     Detect-to-treat:                 development of analysis of Bacilli spores ...
2. EXPERIMENTALLyophilized B. cereus spores, prepared according to literature,16 were supplied by the University of Rhode ...
peaks are observed 657, 758, 1049, 1182, 1428 cm-1, and 1567 cm-1. Several of these peaks have been previouslyassigned bas...
1008 cm-1 peak suggests an LOD of 250 ng. Although this LOD is equivalent to the previous experiment, thisexperiment has a...
REFERENCES1    Jernigan, JA et al. “Bioterrorism-related inhalation anthrax: The first 10 cases reported in the United Sta...
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
Final Report Daad13 02 C 0015 Part5 App G K
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Final Report Daad13 02 C 0015 Part5 App G K

  1. 1. Appendix G Characterization of chemical warfare G-agent hydrolysis products by surface-enhanced Raman spectroscopy Frank Inscore, Alan Gift, Paul Maksymiuk, and Stuart Farquharson* Real-Time Analyzers, 87 Church Street, East Hartford, CT 06108 ABSTRACTThe United States and its allies have been increasingly challenged by terrorism, and since the September 11, 2001 attacksand the war in Afghanistan and Iraq, homeland security has become a national priority. The simplicity in manufacturingchemical warfare agents, the relatively low cost, and previous deployment raises public concern that they may also beused by terrorists or rogue nations. We have been investigating the ability of surface-enhanced Raman spectroscopy(SERS) to detect extremely low concentrations (e.g. part-per-billion) of chemical agents, as might be found in poisonedwater. Since trace quantities of nerve agents can be hydrolyzed in the presence of water, we have expanded our studiesto include such degradation products. Our SERS-active medium consists of silver nanoparticles incorporated into a sol-gel matrix, which is immobilized in a glass capillary. The choice of sol-gel precursor allows controlling hydrophobicity,while the porous silica network offers a unique environment for stabilizing the SERS-active silver particles. Here wepresent the use of these silver-doped sol-gels to selectively enhance the Raman signal of the hydrolyzed products of theG-series nerve agents.Keywords: chemical warfare agent detection, CWA, hydrolysis, SERS, Raman spectroscopy 1. INTRODUCTIONThe potential use of chemical and biological warfare agents by terrorist organizations directed against U.S. military andCoalition forces in the Middle East, and civilians at home, is an issue that has generated considerable concern in the post9/11 era. The ability to counter such attacks, requires recognizing likely deployment scenarios, among which includespoisoning water supplies with chemical warfare agents (CWAs). The G-series nerve agents are a particular concern dueto their extreme toxicity (LD50 man for GB = 25 mg/kg, GD = 5 mg/kg, GF = 5mg/kg ),1 persistence (hydrolysis half-lifeof 1-3 days),2 relatively high solubility (5-25 g/L, see Table 1), and their previous use in Iraq3 and Japan.4 The nerveagents, isopropyl methylphosphonofluoridate (GB), pinacolyl methylphosphonofluoridate (GD), and cyclohexylmethylphosphonofluoridate (GF) initially hydrolyze to isopropyl methylphosphonic acid (IMPA), pinacolylmethylphosphonic acid (PMPA), and cyclohexyl methylphosphonic acid (CMPA), respectively, and subsequently, at amuch slower rate, to a common final, stable product methylphosphonic acid (MPA, see Figure 1).5,6 Clearly any analysisdesigned to detect nerve agents in poisoned water must not only be able to detect µg/L concentrations,7 but also be ableto detect and distinguish the resultant hydrolysis products. In addition, the ability to quantify the relative amounts of theinitial agent and its hydrolysis products would provide a means to estimate when the water supply was poisoned. It isalso worth noting that an analyzer capable of measuring these hydrolysis products at such low concentrations would alsobe valuable in establishing prior presence of nerve agents through soil and groundwater analysis,8,9 verify successfuldestruction during decommissioning operations,5,10,11 and establishing extent of exposure during an attack.12Several technologies have recently been investigated as potential at-site analyzers for chemical agents, as well as theirhydrolysis products.6,13 This includes liquid chromatography combined with mass spectrometry (LC/MS),9,14-17 infraredspectroscopy18,19,20 and Raman spectroscopy (RS).21 However, LC/MS remains a labor intensive technique, infrared islimited by the strong absorption of water which obscures much of the spectrum, while Raman spectroscopy does nothave sufficient sensitivity.21 In the past few years, we and others have explored the potential of surface-enhanced Ramanspectroscopy (SERS) to detect CWAs,22-28 and their degradation products.29 The utility of SERS is based upon theextreme sensitivity of this technique and the ability to identify molecular structure through the abundant vibrationalinformation provided by Raman spectroscopy. SERS employs the interaction of surface plasmon modes of metalparticles with target analytes to increase scattering efficiency by as much as 1 million times.30SPIE-2004-5585 46
  2. 2. In our studies, we have employed metal-doped sol-gels to promote the SERS effect. The porous silica network of thealkoxide sol-gel matrix offers a unique environment for immobilizing and stabilizing SERS-active metal particles of bothsilver and gold.31-34 The choice of metal and Si-alkoxide composition provides a means for chemically selecting thetarget analyte to be enhanced based on charge and polarity. Electropositive silver or electronegative gold particles canselectively enhance the Raman signals of negative or positive chemical species, respectively, while different alkoxides(or combinations of) can be used to select for polar or non-polar molecules. Previously, we used glass vials internallycoated with the SERS-active sol-gel to measure cyanide, HD, VX, and MPA.28 More recently, we have developed glasscapillaries filled with the SERS-active sol-gel that can be attached to a syringe to perform simple and rapid sampleextraction and SERS analysis.35 This paper employs these extractive and SERS-active capillaries to examine the abilityof SERS to measure and distinguish the hydrolysis products of GB, GD, and GF. Both Raman and surface-enhancedRaman spectra are presented along with preliminary vibrational mode assignments. Table 1. Properties of chemical agents and their primary hydrolysis products investigated in the present study.2 Chemical Agent Hydrolysis ½ life Water Solubility at 25°C Sarin (GB) 39 hr (pH 7) completely miscible IMPA stable (can hydrolyze to MPA) 4.8 g/L MPA very stable (resistant to further degradation) >1000 g/L Soman (GD) 45 hr (pH 6.6) 21 g/L (@20°C) PMPA stable (can hydrolyze to MPA) no data Cyclosarin (GF) slower than GB 3.7 g/L CMPA no data (can hydrolyze to MPA) no data H2O O H2O O IMPA 2-propanol + MPAGB HF + P P O F O OH H2O H2 OGD HF + PMPA 2-pinacolyl + MPA O O P P O F O OH H2O H2OGF O + O CMPA cyclohexanol + MPA HF P P O F O OHFigure 1. Hydrolysis pathways for G-Series nerve agents. 2. EXPERIMENTALThe hydrolysis degradation chemicals measured in this study (IMPA, PMPA, CMPA) were obtained as analyticalreference materials from Cerilliant (Round Rock, TX) and used without further purification. MPA and all chemicalsused to prepare the silver-doped sol-gel coated capillaries were acquired from Sigma-Aldrich (St. Louis, MO) and usedas received. For the purpose of safety, samples were prepared in a chemical hood, transferred to a sampling device andsealed prior to being measured. All samples were measured initially by Raman in their pure state at room temperature;MPA as a solid powder, with IMPA, and PMPA as neat liquids. CMPA was obtained in forensic quantities (1 mg/mL inMeOH), and was not amenable to RS studies at these concentration levels.Methanol or water (HPLC grade) was used to prepare solutions of the target chemicals for SERS measurements at aSPIE-2004-5585 47
  3. 3. concentration of 1 mg/mL from solid powders or 0.1% v/v from neat liquids unless noted otherwise. Lowerconcentrations were prepared from these solutions by serial dilution, and all solutions were stored at 10°C until needed.The Raman and SERS spectra of the target chemicals presented here were all measured in capillaries.SERS-active capillaries were prepared using the following general methodology. A silver-doped sol-gel solution,prepared according to previous published procedures from a mixture of two precursor solutions,31 was drawn via asyringe into pre-cleaned 1-mm diameter capillaries. This procedure was modified for the SERS-active capillaries, inparticular by replacing TMOS with an alkoxide mixture composed of tetramethyl orthosilicate (TMOS),octadecyltrimethoxysilane (ODS), and methyltrimethoxysilane (MTMS) at a v/v/v ratio of 1/1/5.A 50 µL sample from each of the prepared analyte solutions was drawn into a SERS-active capillary for measurement.The capillaries were mounted horizontally on an XY positioning stage (Conix Research, Springfield, OR), such that thefocal point of an f/0.7 aspheric lens was positioned just inside the glass wall. The probe optics and fiber optic interfacehave been described previously.35 A Fourier transform Raman spectrometer (Real-Time Analyzers, model IRA-785,East Hartford, CT) equipped with a 785 nm diode laser (Process Instruments Inc. model 785-600, Salt Lake City, UT)and a silicon photo-avalanche detector (Perkin Elmer model C30902S, Stamford, CT) was used to deliver 100 mW ofpower to the SERS and RS samples and generate spectra with 8 cm-1 resolution. 3. RESULTS AND DISCUSSIONThe SERS spectra of chemicals are often different than their Raman spectral counterparts due to the surface interactionsthat can enhance various vibrational modes to different extents. Therefore the Raman spectra were measured andincluded in this study to aid interpretation of the corresponding SERS spectra. The simplest chemical specific to the Gseries nerve agents is methylphosphonic acid, which has been well characterized by IR and Raman spectroscopy,36,37 andsubsequent normal coordinate analysis for assigning the vibrational modes.38 The Raman spectrum of MPA contains 10discernable peaks between 350 and 1650 cm-1 (Figure 2B). Four PO3 bending modes are observed at 408, 462, 491(shoulder) and 504 cm-1. The PC symmetric stretch is the most intense peak observed at 774 cm-1. A CH3 rocking modeoccurs at 892 cm-1 with little intensity, while the PO3 stretching mode produces a peak to 956 cm-1. Two additional CH3and PO3 modes produce peaks at 1004 and 1054 cm-1, also with moderate intensity. The 10th mode in this region is aCH3 bending mode which occurs at 1424 cm-1. A A B BFigure 2. A) SERS and B) Raman spectra of MPA. Figure 3. A) SERS and B) Raman spectra of IMPA.Conditions: A) 0.1 mg/ml in water, TMOS/ODS/MTMS Conditions as in Fig. 2, but: A) 0.1 % v/v in MeOH, B)sol-gel in capillary, 1-min acquisition time. B) solid, 5- neat liquid.min acquisition time.The SERS spectrum of MPA (Figure 2A) is considerably simpler than that of the solid powder Raman spectrum, withweak peaks observed at 469, 521, 958, 1003, 1038, and 1420 cm-1. These SERS spectral peaks can all be assigned to themodes observed at similar frequencies in the Raman spectrum, albeit the 521 and 1038 cm-1 peaks have shiftedsignificantly from the 504 and 1054 cm-1 Raman spectral peaks. The most characteristic SERS spectral peaks are theSPIE-2004-5585 48
  4. 4. intense 756 cm-1 peak and the unique peak at 1300 cm-1. The former peak clearly corresponds to a nearly pure PCsymmetric stretch, while the latter is likely a CH3 twist.The next hydrolysis product studied was isopropyl methylphosphonic acid. Like MPA, both the Raman and SERSspectra of IMPA are dominated by a peak in the 700 cm-1 region, specifically at 728 and 716 cm-1, respectively (Figure3). However, these peaks are not simply a PC stretch, but include a considerable amount of the backbone CPOCC modecreated by the addition of the isopropyl group. Both spectra also contain moderate peaks at 782 and 772 cm-1 that mayalso be PC containing backbone modes, as has been suggested by a theoretical treatment for sarin.39 It is also worthnoting that the Raman spectrum of IMPA is very similar to that of a published spectrum of sarin.21 A number of thepeaks assigned to PO3 modes for MPA have shifted moderately from the Raman to the SERS spectra for IMPA, andincludes the following respective peaks; 510 and 508 cm-1, 938 and 931 cm-1, and 1006 and 1004 cm-1. The latter peaklikely contains significant methyl character. Similarly, the methyl rocking and bending modes observed for MPA arenow at 880 and 874 cm-1, and 1420 and 1416 cm-1 in the respective Raman and SERS spectra of IMPA. Notsurprisingly, the isopropyl group not only increased the intensity of these bands, but also gives rise to a CH deformation,and additional CH3 and CH2 wagging modes, at 1359 and 1349 cm-1, 1390 and 1388 cm-1 and 1453 and 1451 cm-1, in therespective Raman and SERS spectra. The isopropyl group also gives rise to a CC bend at 421 and 424 cm-1, and a CCstretch at 1179 and 1173 cm-1 in the respective Raman and SERS spectra. In the Raman spectrum of IMPA a peak alsoappears at 1104 cm-1 that is characteristic of CO or CC stretches, while in the SERS spectrum a peak appears at 1055cm-1 and is assigned to a PO3 stretch, as was the 1038 cm-1 peak in the MPA SERS spectrum.The Raman spectrum of pinacolyl methylphosphonic acid, like IMPA, contains an increasing amount of CC and CHncharacter (Figure 4B). This includes new peaks at 541, 934, 977, 1212 and 1264 cm-1 that are assigned to a CC3 wag, aCC3 bend, a CCC bend, and two CC stretching modes based on a theoretical treatment for soman.39 The 1300 to 1500cm-1 region again contains a number of CHn bending modes, and the peaks are assigned accordingly. The most obviouschange in the spectrum is that the PC plus backbone mode in the IMPA spectrum has split into two distinct peaks at 732and 761 cm-1. The SERS spectrum for PMPA is dominated by these latter peaks, except that they overlap considerablyproducing a peak centered at 750 cm-1 with a shoulder at 729 cm-1 (Figure 4A). The remaining SERS peaks are evident,but have little intensity, except for the CC3 wag at 543 cm-1, the PO3 stretch at 1037 cm-1, and the CH2 bend at 1444 cm-1.Cyclohexyl methylphosphonic acid was only available as 1 mg/mL in methanol and a Raman spectrum at thisconcentration could not be obtained. The SERS spectrum in many ways is like IMPA with the addition of cyclohexanemodes (Figure 5). This includes peaks at 622, 1023, and 1262 cm-1, that are attributed to ring CC stretching modes, anda peak at 811 cm-1 that is assigned to a ring CH2 bending mode. The most intense peak observed at 747 cm-1 is againassigned to a PC stretch plus backbone mode. A B Wavenumber (cm-1) Figure 4. A) SERS and B) Raman spectra of PMPA. Figure 5. SERS spectrum of CMPA. Conditions as in Conditions as in Fig. 3. Fig. 3, but A) 1 mg/mL in MeOH.In general, the SERS spectra for these alkyl methylphosphonic acids have two common features, the PC stretch producesthe most intense peak, more so than the Raman spectra when compared to the intensity of the other peaks, and the mostSPIE-2004-5585 49
  5. 5. substantial shift in peak frequencies occurs for PO3 modes when compared to the Raman spectra. The increasedintensity of the PC mode suggests that it is perpendicular to the surface, based on previous research that has shown thatmodes couple to the plasmon field more effectively in this orientation.40 The shift in the PO3 frequencies suggests strongsurface interactions through this group. Taken together, the SERS data suggests that these molecules are oriented withthe PO3 group interacting with the silver surface and the methyl group away from the surface. In the case of MPA,especially for the doubly deprotonated anion, the three oxygens could form the base of a tripod on the surface. Thisorientation may become less likely for the other molecules as the alkoxide groups replace the hydroxide group withsurface interaction through the other two oxygens. This change in orientation along with increasing amounts ofbackbone character to the PC stretch could explain the shift and splitting of this mode. Table 2. Tentative vibrational mode assignments for Raman and SERS peaks for VX and its hydrolysis products. MPA IMPA PMPA CMPA Tentative Assignmentsa RS SERS RS SERS RS SERS SERSb 408 421 424 PO3 bend 462c,d 469 441 442 441 PO3 bend 491c 475 PO3 bend 504c 521 510 508 514 495 C-PO3 bend 541e 543 549 C-C3 bend 622 Ring breathing 728 716 732 729sh PC stretch and backbone 774 756 782 772 761 750 747 PC stretch and backbone 799 792 CH bend 811 Ring CH2 880e 874 869e 863 857 CCC bend 892c,d 902 888 896 CH3 rock 934e 929 C-C3 bend c,d 956 958 938 931 PO3 stretch 977e CCC stretch 1004 1003 1006 1004 1015 1000 PO3 or CH3 bend 1023 Ring breathing sym 1054 1038d 1055 1052 1037 1050 PO3 stretch 1079 1073 CCC bend 1104 1116 OC or CC stretch 1143 1132 1150 CC stretch 1179 1173 1212e 1206 CC stretch 1224 1236 1243 CH2 bend or above 1264e 1257 1262 CC stretch 1300 1291 CH3 bend 1359 1349 1355 1347 CH deformation 1374 CHn bend 1390 1388 1390 1394 1393 CH3 rock 1424c,d 1420 1420 1416 1420 1415 1416 CH3 bend (bound to P) 1453 1451 1447 1444 1443 CH2 rocka - Assignment terminology is simplified since assignments refer to multiple molecules. b - no Raman spectrummeasured, c = Ref. 36, d = Ref. 37, e = Ref. 39. 4. CONCLUSIONThe ability to measure and identify the various hydrolysis degradation products with our SERS-active silver-doped sol-gel coated capillaries has been demonstrated. The SERS spectra of these chemicals were somewhat different than theirRaman spectral counterparts, which is attributed to the interaction of these chemicals with the silver. In general, theRaman and SERS spectra for the alkyl methylphosphonic acid hydrolysis products were dominated by one or two peaksbetween 715 and 765 cm-1, which have been assigned to PC stretching modes with varying amounts of backbone modeSPIE-2004-5585 50
  6. 6. contributions. The spectral intensity of this mode and the shift in frequency of the PO3 modes in the SERS spectrasuggest a strong surface interaction for these molecules. It is clear from the present study that the hydrolysis productscan easily be identified as a class by these 700 cm-1 peaks, but quantifying each in a mixture is likely to require chemicalseparations or chemometric approaches. These approaches, as well as measurements to determine the detection limitsand pH dependence of these hydrolysis products are in progress. 5. ACKNOWLEDGMENTSThe authors are grateful for the support of the U.S. Army (DAAD13-02-C-0015, Joint Service Agent Water Monitorprogram), and the National Science Foundation (DMI-0215819), and would like to thank Dr. Steve Christesen forhelpful discussions, and Mr. Chetan Shende for sol-gel chemistry development. 6. REFERENCES1. Committee on Toxicology. Review of Acute Human-Toxicity Estimates for Selected Chemical-Warfare Agents, Nat. Acad. Press (Washington, D.C.) 1997.2. Munro, N.B., Talmage, S.S., Griffin, G.D., Waters, L.C., Watson, A.P., King, J.F., and Hauschild V. “The Sources, Fate, and Toxicity of Chemical Warfare Agent Degradation Products”, Environ. Health Perspect., 107, 933-974 (1999).3. Hoenig, S.L. Handbook of Chemical Warfare and Terrorism, Greenwood Press (Westport, CT) 2002.4. Nozaki, H. and Aikawa, N. “Sarin poisoning in Tokyo subway”, Lancet, 345 1446-1447 (1995).5. Wagner, G. and Yang, Y. “Rapid nucleophilic/oxidative decontamination of chemical warfare agents”, Ind. Eng. Chem. Res., 41, 1925-1928, (2002).6. Creasy, W., Brickhouse, M., Morrissey, K., Stuff, J., Cheicante, R., Ruth, J., Mays, J., Williams, B., O’Connor, R., and Durst, H. “Analysis of chemical weapons decontamination waste from old ton containers from Johnston atoll using multiple analytical methods”, Environ. Sci. Technol., 33, 2157-2162, (1999).7. McKone, T.E., Huey, B.M., Downing, E., and Duffy, L.M., Editors. Strategies to Protect the Health of Deployed U.S. Forces: Detecting, Characterizing, and Documenting Exposures, Nat. Acad. Press (Washington, D.C.) p.207, 1999.8. Johnston, R.L., Hoefler, C.M., Fargo, J.C., and Moberley, B. “The Defense Nuclear Agency’s Chemical/ Biochemical Weapons Agreements Verification Technology Research, Development, Test and Evaluation Program and its Requirements for On-Site Analysis”, AT-ONSITE, 5-8 (1994).9. D’Agustino, P.A, Hancock, J.R., and Provost, L.R. “Determination of sarin, soman and their hydrolysis products in soil by packed capillary liquid chromatography-electrospray mass spectrometry”, J. Chromatography A, 912, 291- 299 (2001).10. Yang, Y., Baker, J., and Ward, J. “Decontamination of chemical warfare agents”, Chem. Rev., 92, 1729-1743 (1992).11. Christesen, S., MacIver, B., Procell, L., Sorrick, D., Carrabba, M., and Bello, J. “Nonintrusive analysis of chemical agent identification sets using a portable fiber-optic Raman spectrometer”, Appl. Spec., 53, 850-855 (1999).12. Hui, D.-M. and Minami, M. “Monitoring of fluorine in urine samples of patients involved in the Tokyo sarin disaster, in connection with the detection of other decomposition products of sarin and the by-products generated during sarin synthesis”, Clin. Chim. Acta, 302, 171-188 (2000).13. “The Chemical Weapons Convention Redefines Analytical Challenge”, Analytical Chemistry News & Features, June 1, 397A (1998).14. Sega, G.A., Tomkins, B.A., and Griest, W.H. “Analysis of methylphosphonic acid, ethyl methylphosphonic acid and isopropyl methylphosphonic acid at low microgram per liter levels in groundwater” J. Chromatography A, 790, 143-152 (1997).15. Creasy, W.R. “Postcolumn Derivatization Liquid Chromatography/Mass Spectrometry for Detection of Chemical- Weapons-Related Compounds” Am. Soc. Mass Spectrom., 10, 440-447 (1999).16. Katagi…, J. Chromatography A, 833, 169-179 (1999).17. Liu, Q., Hu, X., and Xie, J. “Determination of nerve agent degradation products in environmental samples by liquid chromatography–time-of-flight mass spectrometry with electrospray ionization”, Analytica Chimica Acta, 512, 93- 101 (2004).SPIE-2004-5585 51
  7. 7. 18. Hoffland, L.D., Piffath, R.J., and Bouck, J.B. “Spectral signatures of chemical agents and simulants”, Optical Engineering, 24, 982-984, (1985).19. Braue, E.H.J., and Pannella, M.G. “CIRCLE CELL FT-IR Analysis of Chemical Warfare Agents in Aqueous Solutions”, Applied Spectroscopy, 44, 1513-1520, (1990).20. Kanan, S. and Tripp, C. “An infrared study of adsorbed organophosphonates on silica: a prefiltering strategy for the detection of nerve agents on metal oxide sensors”, Langmuir, 17, 2213-2218, (2001).21. Christesen, S.D. “Raman cross sections of chemical agents and simulants”, Appl. Spec., 42, 318-321 (1988).22. Lee, Y. and Farquharson, S. “Rapid chemical agent identification by SERS”, SPIE, 4378, 21-26 (2001).23. Farquharson, S., Maksymiuk, P., Ong, K., and Christesen, S. “Chemical agent identification by surface-enhanced Raman spectroscopy”, SPIE, 4577, 166-173 (2001).24. Spencer, K.M., Sylvia, J., Clauson, S. and Janni, J. “Surface Enhanced Raman as a Water Monitor for Warfare Agents in Water”, SPIE, 4577, 158-165 (2001).25. Premasiri, W., Clarke, R., Londhe, S., and Womble, M. “Determination of cyanide in waste water by low-resolution surface enhanced Raman spectroscopy on sol-gel substrates”, J. Ram. Spec., 32, 919-922 (2001).26. Tessier, P., Christesen, S., Ong, K., Clemente, E., Lenhoff, A., Kaler, E., and Velev, O. “On-line spectroscopic characterization of sodium cyanide with nanostructured Gold surface-enhanced Raman spectroscopy substrates”, App. Spectrosc., 56, 1524-1530 (2002).27. Christesen, S.D., Lochner, M.J., Ellzy, M., Spencer, K.M., Sylvia, J., and Clauson, S. “Surface Enhanced Raman Detection and Identification of Chemical Agents in Water”, 23rd Army Science Conf., Orlando, 2002.28. Farquharson, S., Gift, A., Maksymiuk, P., Inscore, F., Smith, W., Morrisey, K., and Christesen, S. “Chemical agent detection by surface-enhanced Raman spectroscopy”, SPIE, 5269, 16-22 (2004).29. Farquharson, S., Gift, A., Maksymiuk, P., Inscore, F., and Smith, W. “pH dependence of methyl phosphonic acid, dipicolinic acid, and cyanide by surface-enhanced Raman spectroscopy”, SPIE, 5269, 117-125 (2004).30. Weaver, M.J., Farquharson, S., and Tadayyoni, M.A. “Surface-enhancement factors for Raman scattering at silver electrodes”, J. Chem. Phys., 82, 4867-4874 (1985).31. Lee, Y. and Farquharson, S. “SERS Sample Vials Based on Sol-Gel Process for Trace Pesticide Analysis”, SPIE, 4206, 140-146 (2000).32. Farquharson, S. and Lee, Y. “Trace Drug Analysis by Surface-Enhanced Raman Spectroscopy”, SPIE, 4200-16 (2000).33. Lee, Y., Farquharson, S., and Rainey, P.M. “Surface-Enhanced Raman Sensor for Trace Chemical Detection in Water”, SPIE, 3857, 76-84 (1999).34. Lee, Y, Farquharson, S., Kwong, H., and Shahriari, M. “Surface-Enhanced Raman Sensor for Surface-Enhanced Raman Spectroscopy”, SPIE, 3537, 252-260 (1998).35. Farquharson, S., Gift, A., Maksymiuk, P., and Inscore, F. “Rapid dipicolinic acid extraction from Bacillus spores detected by surface-enhanced Raman spectroscopy”, Appl. Spec. 58, 351-354 (2004).36. Nyquist, R. “Vibrational spectroscopic study of (R-PO3)2¯”, J. Mol. Struct., 2, 123-135, (1968).37. Van der Veken, B.J. and Herman, M.A. “Vibrational analysis of methylphosphonic acid and its anions: I. Vibrational spectra”, J. Molec. Struct., 15, 225-236 (1973).38. Van der Veken, B.J. and Herman, M.A. “Vibrational analysis of methylphosphonic acid and its anions: II. Normal coordinate analysis”, J. Molec. Struct., 15, 237-248 (1973).39. Hameka, H. and Jensen, J. “Theoretical prediction of the infrared spectra of nerve agents”, CRDEC-TR-326, 1992.40. Suh, J.S. and Moskovitz, M. “SERS of amino acids and nucleotide bases adsorbed on silver” J. Am. Chem. Soc. 108, 4711-4718 (1986).SPIE-2004-5585 52
  8. 8. Appendix H Surface-enhanced Raman spectra of VX and its hydrolysis products STUART FARQUHARSON,∗ ALAN GIFT, PAUL MAKSYMIUK, AND FRANK INSCORE Real-Time Analyzers, East Hartford, CT 06108Detection of chemical agents as poisons in water supplies, Table I. Hydrolysis half-lifea and water solubilityb for VXnot only requires µg/L sensitivity, but also requires the and its primary hydrolysis products.ability to distinguish their hydrolysis products. We have Chemical Agent Hydrolysis Half-life Water Solubilitybeen investigating the ability of surface-enhanced Raman VX >3 days (pH 7) 150 g/Lspectroscopy (SERS) to detect chemical agents at these EA2192 > 10 x VX ∞ sol.concentrations. Here we expand these studies and present DIASH stable ca. 1000 g/Lthe SERS spectra of the nerve agent VX (ethyl S-2- EMPA >8 days 180 g/Ldiisopropylamino ethyl methylphosphonothioate) and its MPA very stable >1000 g/Lhydrolysis products; ethyl S-2-diisopropylamino a = Ref. 1, b = Ref. 2, c at 25°Cmethylphosphonothioate, 2-(diisopropylamino) ethanethiol,ethyl methylphosphonic acid, and methylphosphonic acid. molecule interacts with the surface plasmon modes of metalVibrational mode assignments for the observed SERS peaks nanoparticles, such as gold or silver,12 which will provide theare also provided. Overall, each of these chemicals necessary sensitivity. Typical enhancements on the order of 1produces a series of peaks between 450 and 900 cm-1 that million times have been reported for MPA,6 and calculatedare sufficiently unique to allow identification. SERS limits of detection (LOD) at 50 to 100 µg/L,8,9 are close to themeasurements were performed in silver-doped sol-gel filled required 10 µg/L LOD for nerve agents in water.13 Thecapillaries that are being developed as part of an extractive expected success of SERS is also based on the unique set ofpoint sensor. Raman spectral peaks due to the specific molecular vibrations of each chemical that will allow unequivocal identification ofINTRODUCTION the nerve agents and their hydrolysis products. Towards fulfilling this second expectation, we have measured the SERS In the post 9/11 era the use of chemical and biological spectra of VX and its hydrolysis products; EA2192, DIASH,warfare agents by terrorist organizations directed against U.S. EMPA, and MPA, and provide preliminary vibrational modeand Coalition forces in Afghanistan and Iraq, as well as assignments. In this study, a silver-doped sol-gel has beencivilians at home is an undeniable possibility. Countering incorporated into a glass capillary to both chemically extractfuture attacks requires recognizing likely deployment scenarios, the target analytes and promote the SERS effect.14among which includes poisoning of water supplies. In thisinstance, the nerve agent ethyl S-2-diisopropylamino ethyl EXPERIMENTALmethylphosphonothioate (VX) is of particular concern, becausein addition to an oral LD50 of 0.012 mg/kg in humans, it is DIASH and EMPA were obtained as analytical referencereasonably soluble (150g/L), and somewhat persistent with a materials from Cerilliant (Round Rock, TX) and used withouthydrolysis half-life greater than 3 days.1 Furthermore, one of further purification. MPA and all chemicals used to prepareits hydrolysis products, ethyl S-2-diisopropylamino the silver-doped sol-gel coated capillaries were acquired frommethylphosphonothioate (EA2192), is considered just as Sigma-Aldrich (St. Louis, MO) and also used as received.deadly, more soluble and more persistent (Table I).2 In fact, For the purpose of safety, all samples were prepared in aVX can hydrolyze according to two different pathways (Fig. 1, chemical hood, transferred to a capillary and sealed prior toReaction Pathways 1 and 2).3,4 In one case, 80% of VX is being measured. The Raman spectra of VX and EA2192 wereconverted to 2-(diisopropylamino) ethanethiol (DIASH), which measured as a pure liquid and a pure solid, respectively at theis stable in water, and ethyl methylphosphonic acid (EMPA), U.S. Army’s Edgewood Chemical Biological Center. Thewhich further hydrolyzes to form methylphosphonic acid Raman spectra of EMPA was measured as a pure liquid, while(MPA) and ethanol. In the other case, 20% of VX is converted both DIASH and MPA were measured near the point ofto EA2192 and ethanol, and as previously indicated, EA2192 saturation as 1 g/mL in HPLC grade water samples. In theeventually hydrolyzes and forms DIASH and MPA. case of surface-enhanced Raman spectral measurements, Previously, we5-8and others 9-11 reported the surface- EMPA was prepared as 0.1% v/v in methanol, DIASH as 1enhanced Raman spectra of VX, EA2192, and MPA as mg/mL in methanol, VX as 1% v/v in water, MPA as 0.1preliminary data to demonstrate the potential of developing a mg/mL in water, and EA2192 as 1 mg/mL in water. VX andportable analyzer capable of measuring µg/L concentrations of EA2192 were measured in 2-ml glass vials internally coatedchemical agents in less than 10 minutes. The expected success with a layer of silver-doped sol-gel (Real-Time Analyzers,of surface-enhanced Raman spectroscopy (SERS) is based on Simple SERS Sample Vials, East Hartford, CT), while MPA,the enormous increase in Raman scattering efficiency when a EMPA, and DIASH were measured in 1-mm diameter glass ∗ Author to whom correspondence should be sent. Applied Spectroscopy, 59, 2005 654
  9. 9. HO O H2O DIASH P Pathway 1 N + EMPA EtOH + OVX OH HS P O OH MPA O H2O P N O S HO H2O EtOH + EA2192 Pathway 2 P N DIASH + MPA O SFIG. 1. Hydrolysis pathways for VX.3,4capillaries filled with silver-doped sol-gel. The latter were RESULTS AND DISCUSSIONprepared according to previously published methods,15 exceptfor the following modification: the alkoxide, tetramethyl The assignment of SERS peaks to vibrational modes is lessorthosilicate (TMOS), was replaced by an alkoxide mixture straightforward than for Raman spectral peaks due to thecomposed of TMOS, methyltrimethoxysilane (MTMS), and metal-to-molecule surface interactions that shift and enhanceoctadecyltrimethoxysilane (ODS) in a v/v/v ratio of 1/1/5. This various modes to different extents. For this reason, the Ramanlatter alkoxide combination produced a more non-polar sol-gel spectra for all of the chemicals investigated were alsothat better extracted the MPA, EMPA, and DIASH from the measured and included in the spectral analysis. The analysissolvent. begins with methyl phosphonic acid, the final hydrolysis Both SERS-active sampling devices were mounted product, since it is the simplest molecule, and the vibrationalhorizontally on an XY positioning stage (Conix Research, modes have been assigned.17-19 This approach providesSpringfield, OR), such that the focal point of an f/0.7 aspheric greater confidence in the assignments of the more complexlens was positioned just inside the glass wall. The probe optics molecules, in particular VX. It should be realized that ethanoland fiber optic interface have previously been described.15 In is also a hydrolysis product, but is SERS-inactive andall cases a 785 nm diode laser (Process Instruments Inc. model consequently not included in this study. Table II summarizes785-600, Salt Lake City, UT) was used to deliver ~100 mW of the assignments of the measured spectral peaks to vibrationalpower to the SERS samples and 100 to 300 mW to the Raman modes for a 1 g/mL aqueous MPA solution. Six of thespectroscopy samples. A Fourier transform Raman possible 24 vibrational modes for this molecule with Csspectrometer (Real-Time Analyzers, model IRA-785) equipped symmetry occur in the solution Raman spectrum between 350with a silicon photo-avalanche detector (Perkin Elmer model and 1650 cm-1 (Fig. 2A). The dominant spectral feature at 763C30902S, Stamford, CT) was used to collect both the Raman cm-1 is assigned to the symmetric PC stretch, which in essenceand SERS spectra at 8 cm-1 resolution and at 5-min and 1-min bonds methyl and phosphate tetrahedral-like structures.acquisition times, respectively, except in the case of the Raman Moderately intense peaks at 444 and 954 cm-1 are assigned tospectra of VX and EA2192. These two measurements, a symmetric PO3 bend and a symmetric PO3 stretch,performed at Aberdeen, used a 785 nm diode laser to deliver respectively. The other three peaks of moderate intensity at100 to 150 mW to the sample. A dispersive spectrometer and a 488, 883, and 1423 cm-1 are assigned to a PO3 bend, a CH3silicon-based CCD detector were used to acquire 1 cm-1 rock, and a CH3 bend, respectively.resolution spectra in 1-min acquisitions (InPhotonics, The SERS spectrum of 0.1 mg/mL MPA is very similar toNorwood, MA).16 the Raman spectrum in general appearance (Fig. 2B), All samples were measured within 1 hour of preparation to dominated by the peak at 756 cm-1, which is again assigned toensure minimum hydrolysis. Only in the case of VX, with the the symmetric PC stretch. This peak has gained intensityshortest hydrolysis half-life, would any significant product relative to all of the other peaks, suggesting that this mode isform in this time frame (< 1%). Furthermore, once the samples perpendicular to the surface, based on previous research thatwere introduced into the vials or capillaries they were measured has shown that modes couple to the plasmon field morewithin 10 minutes. For the vials, this appears to be sufficient effectively in this orientation.20 While shifts in the peaks attime for the sample to diffuse through the sol-gel to the silver 954 and 1423 cm-1 to 958 and 1420 cm-1, respectively, aresurface, as no time dependence was observed for the spectra. minor, shifts in the peaks at 444 and 488 cm-1 to 469 and 521For the capillaries, the sample is drawn through the sol-gel cm-1, respectively, are more substantial. Nevertheless, theseminimizing the amount of diffusion required to reach latter peaks are consistent with Raman spectra of monobasicequilibrium, and again no time dependence was observed for anion of methylphosphonic acid (MPA-), which have beenthe spectra. reported at 462 and 507 cm-1, respectively.18 This is further Applied Spectroscopy, 59, 2005 655
  10. 10. supported by recent pH dependent SERS studies of MPA, that modes are no longer pure PC and can not be oriented show that MPA- is the predominant species at neutral pH and completely perpendicular to the surface. Nevertheless, very low concentrations.8 Two additional peaks appear at 1038 interaction with the silver is still most favored through the and 1300 cm-1. The former has also been reported for the oxygen atoms, which not only shifts the PO2 stretch from 1047 Raman spectrum of MPA- at 1040 cm-1 and has been assigned to 1059 cm-1, but also produces significant enhancement. The to a symmetric PO2 stretch, while the latter peak has been remaining POn and CHn modes shift by less than 10 cm-1 and observed in infrared spectra at 1310 cm-1, and assigned to a are less enhanced by interaction with silver. symmetric CH3 bend.18 Taken together, the shift in the frequency of these PO3 peaks and the increased intensity of the PC mode, the SERS data suggests that MPA is oriented with Raman Intensity (relative) the PO3 group interacting with the silver surface and the methyl B group away from the surface.Raman Intensity (relative) B A 450 650 850 1050 1250 1450 1650 Wavenumber (∆cm-1) FIG. 3. A) Raman and B) SERS spectra of EMPA. Conditions as in Fig. 2, but A) neat liquid, 100 mW of 785 nm, 5-min, B) 0.1 % v/v in MeOH. A The other major hydrolysis product of VX according to Pathway 1 is 2-(diisopropylamino) ethanethiol. The normal 450 650 850 1050 1250 1450 1650 Raman spectrum can be analyzed in terms of an alkanethiol Wavenumber (∆cm-1) and an alkyl substituted tertiary amine. For example, the FIG. 2. A) Raman and B) SERS spectra of MPA. Conditions: A) 1g/mL MPA former chemical type produces a CSH bending mode and two in water, 300 mW of 785 nm, 5-min acquisition time, B) 0.1 mg/ml in water, MTMS/ODS/TMOS sol-gel in glass capillary, 100 mW of 785 nm, 1-min CS stretching modes between 650 and 750 cm-1, and an SH acquisition time. stretching mode at 2570 cm-1.21,22 DIASH contains peaks at 667, 721, 738, and 2569 cm-1 (Fig. 4A), which are assigned to The next simplest hydrolysis product of VX is ethyl these respective modes. The latter chemical type produces methylphosphonic acid, formed according to Pathway 1. The one NC3 breathing mode in the 400-500 cm-1 region and a replacement of a hydroxy with an ethoxy group quickly second breathing mode near 950 cm-1, an NCC bending mode increases the number of predicted vibrational modes to 42, near 570 cm-1, an NC stretching mode near 1200 cm-1, and in decreases the symmetry of the molecule as well as the purity of concert CH bending modes near 740 and 1450 cm-1.23,24 the modes, and adds a CPOCC backbone. In addition to the DIASH contains peaks at 481, 945, 585, 1184, 738, and 1441 appearance of several new peaks, the dominant PC symmetric cm-1, which are assigned to these respective modes. Note that stretch at 763 cm-1 is replaced by a peak at 730 cm-1 in the the assignment of the peak at 738 cm-1 has been assigned to Raman spectrum (Fig. 3A), which is now assigned as a both a CS stretch and a CH bend. Also the most intense peak backbone stretch containing PC and OCC character. The in the spectrum appears at 814 cm-1 and is attributed to a asymmetry of this peak suggests an additional, underlying backbone mode consisting of SC stretching and NC3 breathing peak, which may also be due to a backbone mode. The CH3 modes. The Raman spectrum also contains two low frequency rock and bending modes that occurred for MPA at 883, 1300 peaks at 416 and 435 cm-1 that are attributed to CC or CN (SERS) and 1423 cm-1, are still apparent at 893, 1293 and 1420 bending modes, while more than 12 moderately intense peaks cm-1, while additional CH2 rock, and CH3 and CH2 bending appear between 1000 and 1400 cm-1, which are variously modes occur at 792, 1454 and 1480 cm-1. The MPA PO3 assigned to CC or CN stretches, or CHn bending modes. bending modes at 444 and 488 cm-1 are replaced by PO2 The SERS spectrum of DIASH is dominated by the bending modes at 475 and 503 cm-1, while a new peak at 1047 nitrogen and sulfur containing modes (Fig. 4B), specifically cm-1 is assigned to a PO2 stretch, as was the 1038 cm-1 peak in peaks at 482, 587, 811, and 938 cm-1 can be attributed to the MPA SERS spectrum. The second most intense peak in the modes at similar frequencies in the Raman spectrum. This is Raman spectrum at 1098 cm-1 is characteristic of CO or CC expected for the sulfur modes, since DIASH can couple stretches, and is assigned as such without differentiation. strongly to the silver surface through a deprotonated sulfur. Changes, similar to MPA, occur in the SERS spectrum of Deprotonation is supported by the absence of the 667 and EMPA (Fig. 3B). Again, the PC stretch, or at least the PC 2569 cm-1 peaks assigned to the CSH and SH modes, containing backbone modes, which are now resolved at 727 and respectively, in the SERS spectrum. It is also believed that 746 cm-1, are enhanced the most. However, this enhancement this interaction shifts the CS mode from 738 to 698 cm-1. A relative to the other peaks, is less than for MPA, since the similar shift of 26 cm-1 has been observed for simple Applied Spectroscopy, 59, 2005 656
  11. 11. alkanethiols in the Raman and SERS spectra.25-27 It is also PO2S bend, the OPC stretch, and a PO2 stretch. The believed that the 738 cm-1 peak of moderate intensity in the appearance of the SC stretching mode at 693 cm-1 indicates SERS spectrum of DIASH is the CH bend component of the that sulfur still interacts with silver significantly. But then, the Raman peak. An additional peak occurs in the SERS spectrum absence of the PO2S stretching mode at 1054 cm-1 is difficult at 1032 cm-1 that likely contains some S character. The to explain, and the Raman assignment is therefore, in doubt. enhancement of the two NC3 modes at 482 and 938 cm-1 is somewhat surprising since these modes are sterically excluded by the isopropyl groups from interacting with the surface. Consequently, the enhancement is attributed to a molecular Raman Intensity (relative) orientation with these modes perpendicular to the surface, which is easily attained. BRaman Intensity (relative) B A 450 650 850 1050 1250 1450 1650 Wavenumber (∆cm-1) A FIG. 5. A) Raman and B) SERS spectra of EA2192. Conditions: A) pure solid, 150 mW of 785 nm, 1-min, 1 cm-1, B) 1 mg/mL in water, 100 mW of 785 nm, 1-min in standard SERS vial. 450 650 850 1050 1250 1450 1650 The Raman spectra of VX and EA2192 are surprisingly Wavenumber (∆cm-1) different. This may be attributed, at least to some degree, to FIG. 4. A) Raman and B) SERS spectra of DIASH. Conditions as in Fig. 3, but A) 1g/mL in water, B) 1 mg/mL in MeOH. the fact that VX was measured as a pure liquid, while EA2192 was measured as a solid, the natural states for these two The last hydrolysis product studied in this series is EA2192, chemicals at room temperature. The change in state can and most of the observed Raman peaks can be assigned to the certainly account for the peaks in the VX spectrum to be same modes assigned for the Raman peaks of MPA, EMPA and broader, overlap, and change relative intensity (Fig. 6A). DIASH. Specifically, the Raman peaks at 418, 484, 587, 814, Nevertheless, the following peaks are found at near the same 1132, 1183, 1219, 1306, 1343, 1399, and 1460 cm-1 (Fig. 5A), frequency as the EA2192 peaks; 372, 461, 484, 528, 696, 744, can be assigned to the following DIASH modes; a CC or CN 836, 856, 891, 931, 1015, 1101, 1170, 1214, 1300, 1366, bending mode, an NC3 breathing mode, an NCC bending mode, 1394, 1443, and 1462 cm-1, and are assigned accordingly (see the SCNC3 backbone mode, three NC stretching modes, and Table II). The addition of the ethyl group produces two new four CHn bending modes. Similarly, the peaks at 732 and 1418 peaks at 1101 and 1228 cm-1, which are assigned to an OC cm-1 can be assigned to MPA or EMPA modes; an OPC stretching mode (see EMPA) and a CH2 bending mode. The backbone mode and the CH3 wagging mode of the isolated reappearance of the PC stretching mode at 769 cm-1 suggests methyl group bound to phosphorous. The PS bond connecting that this peak and the 731 cm-1 peak contain significant OPC the MPA and DIASH moieties also produces several new peaks. For example, the peaks at 386, 513, and 1054 cm-1 (the Raman Intensity (relative) latter being the most intense peak in the spectrum) are assigned to SPO bending, PO2S bending and PO2S stretching modes, respectively. The peak at 947 cm-1 is assigned to an NC3 B stretch based on the DIASH spectrum, while a less intense peak at 966 cm-1 is assigned to a PO2 stretch based on the MPA spectrum. It is also worth noting that the peaks at 667 and 2569 cm-1 that were observed for DIASH due to SH modes are absent, as expected. Just as the Raman spectrum of EA2192 is dominated by DIASH peaks, so is the SERS spectrum (Fig. 5B). This A includes peaks at 481, 584, 693, 811, 939, and 1125 cm-1, assigned to an NC3 breathing mode, an NCC bending mode, the shifted CS stretching mode, the SCNC3 backbone mode, 450 650 850 1050 1250 1450 1650 another NC3 stretching mode, and a NCC stretching mode. Wavenumber (∆cm-1) Three additional peaks of significant intensity occur at 526, FIG. 6. A) Raman and B) SERS spectra of VX. Conditions as in Fig. 5, but 735, and 971 cm-1, and are all attributed to phosphate modes, a A) pure liquid, and B) 1% v/v in methanol. Applied Spectroscopy, 59, 2005 657
  12. 12. Table II. Tentative vibrational mode assignments for Raman and SERS peaks for VX and its hydrolysis products MPA EMPA DIASH EA2192 VX Tentative Assignmentsa NR SER NR SER NR SER NR SER NR SER 386 372 376 SPO bend 423 416 418 CC or CN bend 435 CC or CN bend 444b,c 469c 453 456 461 458 POn bend 481d 482 484 481 484 484 NC3 breathing 488b 475 482 499 POn bend 521c 503 505 513 526 528 539 POn(S) bend 585d 587 587 584 NCn bend 645 667 622 PSC bend 667e CSH bend 697f 693 696 CS stretch 730 727 721e 732 735 744 731 PC stretch + backbone (CPOCC) 738d,e 738 CH bend and/or CS stretch 763 756 741sh 746 769 769 PC stretch and/or backbone 792 779 790 CH bend 817 811 814 811 805 SC stretch + NC3 breathing 827 830 831 830 836 820 883b,c 893 891 889 863 856 CH3 bend 904 903 905 891 891 885 OPC stretch / CCN stretch 929 925 946d 938 947 939 931 939 NC3 stretch 954b,c 958 945 966 971 965 POn stretch 1003 1010 1006 1015 1006 POn or CH3 bend 1043 1032 1040 1029 SCCN bend 1038c 1047 1059 1054 PO2(S) stretch 1070 1098 1094 1095 1101 1096 OC or CC stretch 1129 1120 1132 1125 1121 NC stretch 1162 1184d 1205 1183 1170 NC stretch 1224 1219 1214 1220 NC stretch 1228 1237 CH2 bend 1253 1300 1293 1287 1299 1306 1300 1301 CH3 bend 1329 1327 1365 1355 1343 CN bend + CC bend 1366 1365 1366 1397 1399 1394 1400 CH3 bend / NC3 stretch 1423b,c 1420 1420 1416 1418 CH3 bend 1454 1441 1449d 1427 1443 1439 CH2 bend 1451 CHn bend 1480 1461 1459 1460 1464 1462 1462 CHn bend 1493 1547 CH3 benda Assignment terminology is simplified since assignments refer to multiple molecules.b = Ref. 17, c = Ref. 18, d = Refs. 22 and 23, e = Refs. 20 and 21, f = Refs. 24-26character. Most of these assignments are consistent with those isopropyl groups.of a computer predicted Raman spectrum,28 especially since the The SERS spectrum of VX is reasonably similar to theVX modes are significantly delocalized and only the primary Raman spectrum, with corresponding peaks at 376, 458, 539,contributions are listed. The most intense peaks were predicted 731, 939, 1096, 1301, 1439, and 1462 cm-1 readily observedat 455, 546, 713, 759, 762, 880, 1093, 1216, 1414, 1441, and (Fig. 6B). In fact the greatest difference is that the CC and1463 cm-1, and assigned to a PS stretch or CPO bend, PO2SC CHn modes are not enhanced, as expected, and little can bewag, SC stretch, PC stretch, OCC stretch, CC stretch or CH3 said about the orientation of the molecule to the surface, otherrock, OC stretch or CH3 rock, NC stretch, the CH3 bend of the than the PO2S group interacts sufficiently to be enhancedphosphorous methyl group, and two CH bends of the producing the peak at 539 cm-1. It is worth noting that the Applied Spectroscopy, 59, 2005 658
  13. 13. SERS spectra of VX and EA2192 are not that similar. In like to thank Dr. Steve Christesen for helpful discussions, and Mr. Chetan Shende for sol-gel chemistry development.particular, the NC3 modes have little intensity in the VXspectrum. More interestingly, perhaps, is the similarity ____________________________between the EA2192 and DIASH SERS spectra. The principle 1. Y. Yang., Acc. Chem. Res. 32, 109 (1999).difference being the addition of the PC stretching mode at 735 2. Y. Yang, J. Baker and J. Ward, Chem. Rev. 92, 1729 (1992).cm-1. This may simply be due to the fact that both molecules 3. W. Creasy, M. Brickhouse, K. Morrissey, J. Stuff, R. Cheicante, J. Ruth, J. Mays, B. Williams, R. O’Connor, and H. Durst, Environ. Sci. Technol.interact through the sulfur with the metal surface to similar 33, 2157 (1999).extents resulting in similar orientations. However, it is also 4. Q. Liu, X. Hu, and J. Xie, Anal. Chim. Acta 512, 93 (2004).possible that the EA2192 spectrum is of DIASH. This is 5. Y. Lee and S. Farquharson, SPIE-Int. Soc. Opt. Eng. 4378, 21 (2001).possible if EA2192 either hydrolyzed or photodegraded. Since 6. S. Farquharson, P. Maksymiuk, K. Ong, and S. Christesen, SPIE-Int. Soc. Opt. Eng. 4577, 166 (2001).the sample was prepared and measured within 1 hour, and the 7. S. Farquharson, A. Gift, P. Maksymiuk, F. Inscore, W. Smith, K.hydrolysis half-life is on the order of weeks,1 the former Morrisey, and S. Christesen, SPIE-Int. Soc. Opt. Eng. 5269, 16 (2004).explanation seems unlikely. Since the peak intensities did not 8. S. Farquharson, A. Gift, P. Maksymiuk, F. Inscore, W. Smith, SPIE-Int.change during these measurements, photodegradation catalyzed Soc. Opt. Eng. 5269, 117 (2004). 9. K. M. Spencer, J. Sylvia, S. Clauson, and J. Janni, SPIE-Int. Soc. Opt.by silver also seems unlikely. Further experiments are Eng. 4577, 158 (2001).required to clarify this point. 10. P. Tessier, S. Christesen, K. Ong, E. Clemente, A. Lenhoff, E. Kaler, and O. Velev, Appl. Spectrosc. 56, 1524 (2002).CONCLUSION 11. S. D. Christesen, M. J. Lochner, M. Ellzy, K. M. Spencer, J. Sylvia, and S. Clauson, 23rd Army Science Conference, Orlando (2002). 12. D. L. Jeanmaire and R. P. Van Duyne, J. Electroanal. Chem. 84, 1 (1977). We have reported the SERS spectra of VX and its hydrolysis 13. T. E. McKone, B. M. Huey, E. Downing, and L. M. Duffy, Strategies toproducts, EA2192, DIASH, EMPA, and MPA. Tentative Protect the Health of Deployed U.S. Forces: Detecting, Characterizing,vibrational mode assignments for the observed SERS peaks and Documenting Exposures (National Academy Press, Washington, D.C., 2000) p.207.have also been provided. This was accomplished with the aid 14. S. Farquharson and P. Maksymiuk, Appl. Spectrosc. 57, 479 (2003).of the corresponding Raman spectra for these chemicals. 15. S. Farquharson, A. Gift, P. Maksymiuk, and F. Inscore, Appl. Spectrosc.Overall the SERS spectra consisted of unique peaks at 58, 351 (2004).approximately 460, 530, 730, 760, and 890 cm-1, assigned to 16. S. Christesen, B. MacIver, L. Procell, D. Sorrick, M. Carrabba, and J. Bello, Appl. Spectrosc. 53, 850 (1999).POnX (X= O or S) and PC and PS backbone modes. The 17. R. A. Nyquist, J. Mol. Struct. 2, 123 (1968).contribution of these modes had sufficient variability that each 18. B. J. Van Der Veken and M. A. Herman, J. Mol. Struct. 15, 225 (1973).chemical could be uniquely identified by its SERS spectrum in 19. B. J. Van Der Veken and M. A. Herman, J. Mol. Struct. 15, 237 (1973).this low frequency region. However, quantifying each of these 20. J. S. Suh and M. Moskovitz, J. Am. Chem. Soc. 108, 4711 (1986). 21. M. Hayashi, Y. Shiro, H. Murata, Bull. Chem. Soc. Jpn. 39, 112 (1966).chemicals in an aqueous mixture may require chemical 22. T. Torgrimsen and P. Kleboe, Acta Chem. Scand. 24, 1139 (1970).separations or chemometric approaches. Such approaches, 23. C. Crocker and P. L. Goggin, J. Chem. Soc. Dalton Trans. 5, 388 (1978).along with establishing detection limits and pH dependence for 24. C. Gobin, P. Marteau, and J.-P. Petitet, Spectrochim. Acta 60, 329 (2004).these chemicals are currently being pursued. 25. T. H. Joo, K. Kim, and M. S. Kim, J. Phys. Chem. 90, 5816 (1986). 26. C. H. Kwon, D. W. Boo, H. J. Hwang, and M. S. Kim, J. Phys. Chem. B 103, 9610 (1999).ACKNOWLEDGMENTS 27. A. Kudelski, Langmuir 19, 3805 (2003). 28. H. Hameka and J. Jensen, ERDEC-TR-065 (1993).The authors are grateful for the support of the U.S. Army (DAAD13-02-C-0015, Joint Service Agent Water Monitor program). The authors would also Applied Spectroscopy, 59, 2005 659
  14. 14. Appendix I Detect-to-treat: development of analysis of Bacilli spores in nasal mucus by surfaced-enhanced Raman spectroscopy Frank E. Inscore, Alan D. Gift, and Stuart Farquharson* Real-Time Analyzers, Inc., East Hartford, Connecticut 06108 ABSTRACTAs the war on terrorism in Afghanistan and Iraq continue, future attacks both abroad and in the U.S.A. are expected. Inan effort to aid civilian and military personnel, we have been investigating the potential of using a surface-enhancedRaman spectroscopy (SERS) sampling device to detect Bacillus anthracis spores in nasal swab samples. Such a devicewould be extremely beneficial to medical responders and management in assessing the extent of a bioterrorist attack andmaking detect-to-treat decisions. The disposable sample device consists of a glass capillary filled with a silver-dopedsol-gel that is capable of extracting dipicolinic acid (DPA), a chemical signature of Bacilli, and generating SERS spectra.The sampling device and preliminary measurements of DPA extracted from spores and nasal mucus will be presented. Keywords: Dipicolinic acid; Bacillus spores; Anthrax; Surface-enhanced Raman spectroscopy. 1. INTRODUCTIONIn the autumn of 2001 the threat of conventional suicide-bombing terrorism and bioterrorism within the United Statesbecame a grave reality. Consequently, future terrorist attacks both at home and abroad against civilian and militarypersonnel alike are undeniable possibilities. In the case of using anthrax causing spores as a terrorist weapon, much waslearned from the distribution of endospores through the U.S. postal system.1-6 For example, it was established thatdetection of exposure within the first few days allowed successful treatment of victims using Ciproflaxin, deoxycyclineand/or penicillin G procaine.5 However, the National Naval Medical Center who processed 3,936 nasal swab samplesfrom the Capitol Hill, DC and Brentwood, NJ postal facility employees, required 2-3 days of growing microorganisms inculture media to establish that all but six employees were uninfected.6 The remaining six employees were alsouninfected, but the samples required further analysis. This process was reported as “extremely time-consuming andlabor-intensive”. This re-emphasizes the much stated need for methods to rapidly detect Bacillus anthracis spores sothat emergency responders and management can assess the extent of the event and make detect-to-treat decisions.Nevertheless, the challenges are formidable considering that the Center for Disease Control (CDC) estimates thatinhalation of 10,000 anthracis endospores or 100 nanograms will be lethal to 50% of an exposed population (LD50).7Although polymerase chain reactions (PCR)8,9 and immunoassays5,10,11 have been developed to augment or replace thestandard laboratory method of culture growth, they still have significant limitations. PCR still requires hours to performand each analyzer is limited to the number of samples that can be measured, while the latest immunoassays designed todetect the response of immunoglobulin G to the protective antigen of B. anthracis are only 80% specific and require atleast 10 days after infection to be detected.5As an alternative to these methods, several researchers have been investigating the analysis of calcium dipicolinate(CaDPA) as a B. anthracis signature.12-14 This approach is viable because only spore forming bacteria contain CaDPA,and the most common, potentially interfering spores, such as pollen and mold spores, do not. It has been long knownthat Raman spectra of Bacilli spores are dominated by bands associated with CaDPA15 and that these spectra mayprovide a suitable anthrax signature at the genus level.16 With this in mind, we have been investigating the potential ofusing a surface-enhanced Raman spectroscopy (SERS) sampling device to detect spores in nasal swab samples. Thedesign, intended for medical responders, employs disposable SERS-active capillaries (one per analysis) that can beeasily analyzed using a portable Raman analyzer.17 This approach is based on our previous SERS measurements ofdipicolinic acid (DPA), the acid of CaDPA, both in water18,19 and extracted from B. cereus spores.20SPIE-5585 2004 53
  15. 15. 2. EXPERIMENTALLyophilized B. cereus spores, prepared according to literature,16 were supplied by the University of Rhode Island andused as received. Dipicolinic acid (2,6-pyridinedicarboxylic acid), dodecylamine (DDA), and all chemicals used toprepare the silver-doped sol-gel coated capillaries were obtained from Sigma-Aldrich (Milwaukee, WI) and used withoutfurther purification. The SERS-active capillaries were prepared according to previous published procedures for theSimple SERS Sample Vials using a silver amine precursor and an alkoxide precursor with the following modifications.17The alkoxide precursor employed a combination of methyltrimethoxysilane (MTMS) and tetramethyl orthosilicate(TMOS) in a v/v ratio of 6/1, which was mixed with the amine precursor in a v/v ratio of 1/1. Approximately 15 microLof the mixed precursors were then drawn into a 1-mm diameter glass capillary coating a 15-mm length. After sol-gelformation, the incorporated silver ions were reduced with dilute sodium borohydride.The serial diluted samples of DPA were prepared in HPLC grade water. B. cereus samples were prepared using ~0.1mm3 particles with a typical mass of 0.1 mg. The sample masses were consistent with a previous determination of sporedensity at 0.081 g/mL that indicated a high degree of entrained air. These particles were carefully divided into 3 or 10equal specks prior to the addition of DDA or nasal mucus (see RESULTS AND DISCUSSION). DPA or B. cereusspores were artificially added to nasal mucus samples that were collected in 20 mL glass vials by expulsion. The DPAin mucus samples were prepared by mixing equal volumes of 1mg/mL DPA in water and mucus. The B. cereus inmucus samples were prepared by adding a finely diced 0.1 mg spore sample to 100 microL of mucus.For each of the spore samples, either specks or 100 microL of spore containing mucus, 100 µL drop of a 50 mM DDAsolution in ethanol, pre-heated to 78 oC, was added and allowed to digest the spore coat for 1 minute. The resultantsolutions, as were the DPA in water samples, were drawn into SERS-active capillaries for analysis. This wasaccomplished by mounting the capillaries horizontally to an XY positioning stage (Conix Research, Springfield, OR)just inside the focal point of an f/0.7 aspheric lens. The probe optics and fiber optic interface have been describedpreviously.20 A Fourier transform Raman spectrometer (Real-Time Analyzers, model IRA-785, East Hartford, CT)equipped with a 785 nm diode laser (Process Instruments Inc. model 785-600, Salt Lake City, UT) and a silicon photo-avalanche detector (Perkin Elmer model C30902S, Stamford, CT) was used to deliver 100 mW of power to the SERSsamples and generate spectra with 8 cm-1 resolution. 3. RESULTS AND DISCUSSIONPreviously we reported SERS spectra of dipicolinic acid at a series of concentrations obtained in 2-mL glass vialsinternally coated with a silver-doped sol-gel as the SERS-active media.19 This included samples as low as 1 mg/L using100 mW of 785 nm and 1-min acquisition time. For this concentration the signal was barely discernable above the noisefor the 1008 cm-1 peak (signal-to-noise, S/N =5.6), and alimit of detection (LOD, defined as a S/N of 3) wasestimated just below the measured value at 540 microg/L.One limitation of these vials is that the sample must Adiffuse through the porous sol-gel to the silver surface forSERS to occur. Since this might limit sensitivity orrequire allowance for diffusion, we have developed sol-gelfilled capillaries. A syringe allows drawing the samplethrough the sol-gel in a couple of seconds forcing analyte- Bto-surface interactions. In an effort to establish that theseSERS-active capillaries provide better sensitivity, a set ofserially diluted solutions of DPA in HPLC grade waterwere prepared and measured.Figure 1 shows that, as desired, a significantly better DPASERS spectrum was obtained for 1 mg/L using the Figure 1. SERS spectra of DPA in water at A) 1 mg/Lcapillaries rather than the vials. In fact 10 microg/L and B) 10 microg/L (100 pg in 10 microL sample) usingsamples repeatedly produced spectra (Figure 1B). Intense the SERS-active capillaries, 100 mW of 785 nm and 1-peaks are observed at 815, 1008, and 1382 cm-1, moderate min acquisition time.SPIE-5585 2004 54
  16. 16. peaks are observed 657, 758, 1049, 1182, 1428 cm-1, and 1567 cm-1. Several of these peaks have been previouslyassigned based on the Raman spectrum of DPA as follows:15,16,20 the 1008 cm-1 peak to the symmetric ring stretch, the1382 cm-1 peak to the O-C-O symmetric stretch, the 1428 cm-1 peak to the symmetric ring C-H bend, and the 1567 cm-1peak to the asymmetric O-C-O stretch. The 10 microg/L sample was used to estimate an LOD of 1 microg/L (S/Nequaled 33 for the 1008 cm-1 peak). This was consistent with the fact that attempted measurements of 1 microg/Lsamples did yield spectra, but not in every case. It is also worth noting that only 10 microL samples were used togenerate the spectra, or in the case of the 10 microg/L sample, 100 pg of DPA.Previously, the SERS-active capillaries were used to measure DPA extracted from ~10 microg of Bacillus cereus spores,and preliminary spectra were reported.20 The procedure is described here (Figure 2). Three 0.1 mg samples of B.cereuswere weighed and then each diced into ~ 10 equal parts (~10 microg or 10 million spores), which allowedperforming 30 measurements. To each particle 100 microL of 50 mM DDA in ethanol at 78 oC was added. After 1minute the solution was drawn into a SER-active capillary, which was then mounted above a laser excitation beam suchthat the surface-enhanced Raman spectrum could be acquired. Figure 2E shows a representative spectrum for one ofthese capillaries using a 1-min acquisition time. The primary DPA peaks at 657 cm-1, 815 cm-1, 1008 cm-1, 1382 cm-1,and 1428 cm-1 are easily seen. Again, the S/N of the 1008 cm-1 peak, which was measured as 120, was used to estimatean LOD of 250 ng or 25,000 B. cereus spores in 100 microL DDA. Since it is known that B. cereus spores contain 10-15% DPA (as calcium dipicolinate),21 and that the majority of the DPA is extracted by hot DDA,14 this LOD can becompared to DPA in water. Accordingly, the 10 microg of spores per 100 microL DDA is approximately equivalent to10 mg of DPA per L water, and consequently the LOD is equivalent to 250 microg/L, which is considerably lesssensitive than the 10 microg/L measured for DPA in water. A B E C F Figure 2. Sample preparation includes A) three initial 0.1 mg B. cereus spore samples, B) addition of 100 microL 78 o C 50 mM DDA to ~10 microg portion, C) drawing 10 microL into SERS-active capillary, and D) mounting D capillary in Raman sample compartment. E) SERS spectrum of representative 10 microg sample using 150 mW of 785 nm and 1-min acquisition time. F) SERS spectrum of representative 2 microg sample using 100 mW of 785 nm and 1-min acquisition time.In an effort to measure fewer spores, anhydrous ether was used to disperse spores on a surface to the point of beinginvisible to the unaided eye. In this series of experiments a 0.1 mg B. cereus sample was divided into three nearequivalent specks. To each speck 600 microL of ether was added and allowed to dry. The dispersed spores and etherproduced a solvent ring ~5 cm in diameter with a significant portion of the spores at the edge. A non-cotton swab wasused to collect the residual spores in the center 1/3rd of this area. The swab was added to a vial containing 100 microL of50 mM DDA in ethanol heated to 78 oC. After 1-min, ~ 10 microL of this solution was extracted into a SERS-activecapillary and measured as before. The peaks in the SERS spectrum, acquired in 1-min, are ~ 1/5th the intensity of thosein the previous experiment, suggesting a collected sample of ~2 microg (Figure 2F). The measured S/N of 25 for theSPIE-5585 2004 55
  17. 17. 1008 cm-1 peak suggests an LOD of 250 ng. Although this LOD is equivalent to the previous experiment, thisexperiment has at least lowered the measured amount of spores by a factor of 5. In either case, comparison to themeasurement of 10 microg/L DPA, suggests that these procedures include considerable losses in extracting the DPAfrom the spores and transferring it to the silver surface. Conversely, if the efficiency of these procedures can beimproved then 1 ng or 100 spores should be able to be detected.In an effort to establish baseline sensitivity for sporescontained in nasal mucus, several samples were preparedand measured. Although nasal mucus is mostly water, itcontains sulfate, sugars, proteins (including albumin), Aprotective enzymes and phagocytes, as well as mucin, aglycoprotein. Consequently, the first samples consistedonly of DPA added to nasal mucus to evaluate thepotential chemical and spectral interferences that couldresult from this matrix. Approximately 10 microL of a 0.5 Bmg/mL DPA in a 50/50 mucus/water mixture was drawninto a SERS-active capillary without any pretreatment andmeasured. Although the matrix produced a significantoffset of the baseline, the primary, characteristic spectralpeaks of DPA were easily observed (Figure 3). Figure 3. SERS spectra of A) 0.5 mg/mL DPA in a 50/50Next finely divided specks of B. cereus were added to nasal mucus/water mixture and B) 1 mg/ml DPA in HPLCnasal mucus, thoroughly mixed, and treated with hot DDA. water for comparison. Conditions as in Fig. 1, but A) 5-Again 10 microL samples were drawn into the SERS- min.active capillaries and measured. Unfortunately, no peakswere observed, even when the sample was kept at 78 oC for 10 minutes. Several possibilities may explain this result. Itis possible that chemicals within mucus 1) react with or coat the spores protecting them from digestion by the DDA, 2)react with DDA making it ineffective in digesting the spores, 3) effectively clog the sol-gels preventing released DPAfrom reaching the silver particles, 4) react with the silver particles and deactivate their Raman signal enhancingproperties, 5) react with DPA making it unavailable for measurement, or 6) any combination of these possibilities. Thesuccessful measurement of DPA in nasal mucus suggests that possibilities 3 and 4 are not the major reason for beingunable to detect DPA extracted from spores contained in mucus. Experiments are currently being designed and tested todetermine which of these possibilities is hindering the measurement. 4. CONCLUSIONTowards the goal of developing a simple SERS-active sample device to measure Bacillus anthracis spores in nasalmucus, we have measured 100 pg dipicolinic acid in a 10 microL water sample, suggesting that as few as 100 sporescould be measured. However, only 0.2 microg of B. cereus spores in a 10 microL sample were measured loweringexpectations to 20,000 spores. Furthermore, SERS spectra were not obtained for B. cereus spores artificially added tonasal mucus. Current research is aimed at determining the factors that hindered this last measurement, and at developingthe appropriate separation methods to overcome this limitation. However, it is worth noting that the presented methodcan be used to detect spores on surfaces, and may have value in determining the extent of facility contamination. ACKNOWLEDGEMENTSThe authors are grateful for the support of the National Science Foundation (DMI-0296116 and DMI-0215819) and theU.S. Army (DAAD13-02-C-0015, Joint Service Agent Water Monitor program). The authors are indebted to ChetanShende for preparing the SERS-active capillaries. The authors would also like to thank James Gillespie, Nicholas Fell,and Augustus Fountain for providing important background information, and Professor Jay Sperry of the University ofRhode Island for supplying B. cereus spores.SPIE-5585 2004 56
  18. 18. REFERENCES1 Jernigan, JA et al. “Bioterrorism-related inhalation anthrax: The first 10 cases reported in the United States”, Emerg. Infect. Dis. 6, 933-944 (2001).2 Klietmann, WF, and KL Ruoff “Bioterrorism: implications for the clinical microbiologist,” Clin. Microbiol. Rev. 14, 364-381 (2001).3 Rotz, LD, AS Khan, SR Lillibridge, SM Ostroff, and JM Hughes, “Public health assessment of potential biological terrorism agents,” Emerg. Infect. Dis. 8, 225-230 (2002).4 Dewan, PK et al. “Inhalational Anthrax Outbreak among Postal Workers, Washington, D.C., 2001,” Emerg. Infect. Dis. 8, 1066-1072 (2002).5 Bell DM, PE Kozarsky, D. Stephens, “Clinical issues in the prophylaxis, diagnosis, and treatment of anthrax,” Emerg. Infect. Dis. 8, 222-225 (2002);6 Kiratisin, P et al. “Large-scale screening of nasal swabs for Bacillus anthracis: Descriptive summary and discussion of the National Institute of Health’s experience”, J. Clin. Microbio., 3012-3016 (2002)7 Ingelsby TV, et al. “Anthrax as a biological weapon, 2002: Updated recommendations for management,” J. Amer. Med. Ass. 287, 2236-52 (2002)8 Glick, BR, and JJ Pasternak, Molecular biology: Principles and Applications of Recombinant DNA, ASM Press, Wash. D.C. (1994).9 Bell CA, Uhl JR, Hadfield TL, David JC, Meyer RF, Smith TF, Cockerill III FR, ”Detection of Bacillus Anthracis DNA by LightCycler PCR” J. Clin. Microbiol. 40, 2897 (2002).10 Gatto-Menking DL, Yu H, Bruno JG, Goode MT, Miller M, Zulich AW “Sensitive detection of biotoxoids and bacterial spores using an immunomagnetic electrochemiluminescence sensor” Biosens. Bioelectron. 10, 501-507 (1995).11 Quinlan JJ and Foegeding PM, J. Rapid Methods Automation Microbiol. 6: 1(1998)12 Nudelman R, Bronk BV, Efrima S “Fluorescence Emission Derived from Dipicolinate Acid, its Sodium, and its Calcium Salts” App. Spectrosc. 54, 445-449 (2000)13 Rosen DL, Sharpless C, and McBrown LB “Bacterial spore detection and determination by use of terbium dipicolinate photoluminescence,” Anal. Chem. 69, 1082-1085 (1997)14 Pellegrino PM, Fell Jr NF, and Gillespie JB “Enhanced spore detection using dipicolinate extraction techniques,” Anal. Chim. Acta 455, 167-177 (2002)15 Woodruff WH, Spiro TG, and Gilvarg C “Raman Spectroscopy In Vivo: Evidence on the Structure of Dipicolinate in Intact Spores of Bacillus Megaterium,” Biochem. Biophys. Res. Commun. 58, 197 (1974)16 Ghiamati E, Manoharan R, Nelson WH, and Sperry JF “UV Resonance Raman spectra of Bacillus spores” Appl. Spectrosc. 46, 357- 364 (1992)17 Farquharson, S and P Maksymiuk, “Simultaneous chemical separation and surface-enhancement Raman spectral detection using silver-doped sol-gels,” Appl. Spectrosc., 57, 479-482 (2003)18 Farquharson S, Smith WW, Elliott S and Sperry JF “Rapid biological agent identification by surface-enhanced Raman spectroscopy,” SPIE 3855: 110-116 (1999)19 Farquharson, S, A Gift, P Maksymiuk, F Inscore, and W Smith, “pH dependence of methyl phosphonic acid, dipicolinic acid, and cyanide by surface-enhanced Raman spectroscopy”, SPIE 5269, 117-125 (2004)20 Farquharson, S., A. Gift, P. Maksymiuk, and F. Inscore, “Rapid dipicolinic acid extraction from Bacillus spores detected by surface-enhanced Raman spectroscopy”, Appl. Spectrosc., 58, 351- 354 (2004).21 F.W. Janssen, A.J. Lund, and L.E. Anderson, Science, 127, 26, (1958).SPIE-5585 2004 57

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