“sf” RTA P2001#05 DOD SBIR Phase II Contract Number DAAD13-02-C-0015   Final Report                  December 2005        ...
Appendix A           Rapid chemical agent identification by surface-enhanced Raman                                    spec...
absorption of water would obscure most other chemicals present. Nevertheless, efforts to overcome these limitations havebe...
2                                                                 10                                                      ...
2-Chloroethyl ethyl sulfide, a blister agent simulant, has a chemical structure similar to the mustard gas (Cl-CH2-CH2-S-C...
4. CONCLUSIONSWe have successfully measured the SER spectra of chemical agent simulants: dimethyl metylphosphonate and 2-c...
14   Tseng, C.-H., Mann C.K., and Vickers T.J., “Determination of Organics on Metal Surfaces by Raman Spectroscopy”,   App...
Appendix B      Detection of bioagent signatures: A comparison of electrolytic and metal-                   doped sol-gel ...
Raman spectroscopy has a rich history of investigating biochemical and biological processes.7 Some of the earliest laser-R...
demonstrated reversible and reproducible measurements of p-aminobenzoic acid (PABA) in a flow through system. Here weinves...
electrode surface for E-SERS, through the vial glass wall and into the silver-doped sol-gel film for SG-SERS, or through t...
contributions from signals or baseline offsets. The measurement error is given as S±RMS, and for adenine this equals 2.34%...
A              A                                                          B              B                                ...
The SG-SER spectrum of AMP is considerably different. The adenine bands virtually disappear, except for the two primaryban...
A               B                                                                                       OPO               ...
A final comparison was made between the two SER methods by measuring dipicolinic acid (DPA). This chemical may beinvaluabl...
intensities for RNA extracted from E. coli are very similar to those measured by normal Raman spectroscopy. Although there...
6. REFERENCES1.     Roberts, W.L and Rainey, P.M., Clin. Chem., 39, 1872-1877 (1993).2.     Pasechnik, V.A., C.C. Shone, a...
Appendix C                                       Chemical agent identification by                                    surfa...
Table 1. Chemical Agent Structures, Hydrolysis Half-lives, and JSAWM Thresholds.            Agent                    Short...
Nevertheless, MPA was readily observed for a 10 ppm sample, with an estimated detection limit of 0.4 ppm (based on asignal...
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
Final Report Daad13 02 C 0015 Part5 App A F
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Final Report Daad13 02 C 0015 Part5 App A F

  1. 1. “sf” RTA P2001#05 DOD SBIR Phase II Contract Number DAAD13-02-C-0015 Final Report December 2005 AppendicesA Lee, Y.H. and S. Farquharson, "Rapid chemical agent identification by surface-enhanced Raman spectroscopy ", SPIE, 4378, 21-26 (2001).B Farquharson, S., W.W. Smith, Y.H. Lee, S. Elliott and J. F. Sperry, "Detection of bioagent signatures: A comparison of electrolytic and metal-doped sol-gel surface-enhanced Raman media", SPIE, 4575, 62-72 (2002).C Farquharson, S., P. Maksymiuk, K. Ong and S.D. Christesen, "Chemical agent identification by surface- enhanced Raman spectroscopy", SPIE, 4577, 166-173 (2002).D Farquharson, S., A. Gift, P. Maksymiuk, and F. Inscore, “Rapid dipicolinic acid extraction from Bacillus spores detected by surface-enhanced Raman spectroscopy”, Applied Spectroscopy, 58, 351- 354 (2004).E 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)F Farquharson, S, A Gift, P Maksymiuk, F Inscore, W Smith, K Morrisey and SD Christesen, “Chemical agent detection by surface-enhanced Raman spectroscopy”, SPIE, 5269,16-22 (2004).G Inscore, F, A Gift, P Maksymiuk, and S Farquharson, “Characterization of chemical warfare G-agent hydrolysis products by surface-enhanced Raman spectroscopy”, SPIE, 5585, 46-52 (2005).H Farquharson, S, A Gift, P Maksymiuk, and F Inscore, “Surface-enhanced Raman spectra of VX and its hydrolysis products”, Applied Spectroscopy, 59, 654-660 (2005).I Inscore, FE, AD Gift, Stuart Farquharson, “Detect-to-treat: development of analysis of Bacilli spores in nasal mucus by surface-enhanced Raman spectroscopy”, SPIE, 5585, 53-57 (2005).J Farquharson, S, W Smith, C Brouillette, and F Inscore, “Detecting Bacillus spores by Raman and surface- enhanced Raman (SERS) spectroscopy”, Spectroscopy, June supplement, 8-15 (2005).K Inscore, F, A Gift, P Maksymiuk, JF Sperry, and S Farquharson, “Identifying surfaces contaminated with Bacillus spores using surface-enhanced Raman spectroscopy to detect dipicolinic acid”, in Applications of Surface-Enhanced Raman Spectroscopy, Ed. S Farquharson, CRC Press, Boca Raton, FL, acceptedL Christesen, S, K Spencer, S Farquharson, F Inscore, K Gonser, J Guicheteau “Surface-enhanced Raman detection of chemical agents in water”, in Applications of Surface-Enhanced Raman Spectroscopy, Ed. S Farquharson, CRC Press, Boca Raton, FL, accepted.M Farquharson, S, F Inscore, S Christesen “Detecting chemical agents and their hydrolysis products in water”, in Surface-Enhanced Raman Scattering – Physics and Applications Eds. K Kneipp, M Moskovitz, and H Kneipp, Springer, accepted.N Inscore, F, S Farquharson, “Detecting hydrolysis products of blister agents in water by surface-enhanced Raman spectroscopy”, SPIE, 5993, 19-23 (2005).O Inscore, F, P Maksymiuk, S Farquharson, “Surface-enhanced Raman spectroscopic characterization of the chemical warfare agent vesicant HD and related mono-sulfides”, JRS, in preparation.P ROC curve data from measurements of CN, HD, and VX at the US Army’s Edgewood ChemBio Center. 74
  2. 2. Appendix A Rapid chemical agent identification by surface-enhanced Raman spectroscopy Yuan-Hsiang Lee and Stuart Farquharson* Real-Time Analyzers, 87 Church Street, East Hartford, CT 06108 ABSTRACTAlthough the Chemical Weapons Convention prohibits the development, production, stockpiling, and use of chemicalwarfare agents (CWAs), the use of these agents persists due to their low cost, simplicity in manufacturing and ease ofdeployment. These attributes make these weapons especially attractive to low technology countries and terrorists. Themilitary and the public at large require portable, fast, sensitive, and accurate analyzers to provide early warning of the use ofchemical weapons. Traditional laboratory analyzers such as the combination of gas chromatography and mass spectrometry,although sensitive and accurate, are large and require up to an hour per analysis. New, chemical specific analyzers, such asimmunoassays and molecular recognition sensors, are portable, fast, and sensitive, but are plagued by false-positives(response to interferents). To overcome these limitations, we have been investigating the potential of surface-enhancedRaman spectroscopy (SERS) to identify and quantify chemical warfare agents in either the gas or liquid phase. The approachis based on the extreme sensitivity of SERS demonstrated by single molecule detection, a new SERS material that we havedeveloped to allow reproducible and reversible measurements, and the molecular specific information provided by Ramanspectroscopy. Here we present SER spectra of chemical agent simulants in both the liquid and gas phase, as well as CWAhydrolysis products.Keywords: Chemical warfare agent, simulant, hydrolysis product, SERS, Raman spectroscopy, sol-gels, vapor 1. INTRODUCTIONChemical warfare has been banned since the 1925 Geneva Protocol, yet the use of chemical agents has persisted.1 This canbe attributed to the simplicity in manufacturing, ease of deployment, and the relatively low cost of chemical warfare agents(CWAs). These attributes make these weapons especially attractive to low technology countries and terrorists. Well knownexamples include the large-scale use of tabun (GA) during the Iran-Iraq war (1984-1948),2 and the release of sarin (GB) inthe Tokyo subway in 1995. The latter is the first documented terrorist use of a chemical weapon.3,4 This ever-present threatwas again substantiated by the United Nations Special Commissions report that described Iraq’s facilities for nerve agents,anthrax and small pox production.5-7 These uses of chemical weapons have motivated the development of fast and accurateanalytical techniques to warn soldiers and the public at large. The development of these analytical techniques is challenging,in that these techniques must not only measure extremely low concentrations quickly (microgram/liter in < 1minute), butmust also be capable of measuring both gas phase and liquid phase to be effective. The latter is required since chemicalagents can also be used to "poison" water supplies.8,9The earliest technologies developed for CWA detection were based on electrochemical, ionization, or colorimetric analysis(e.g. phosgene tape). Although these analyzers were easy to use, they were not generally agent specific and suffered fromfalse-positives.1 More traditional laboratory methods have also been investigated, and in particular, combined gaschromatography and mass spectrometry (GC/MS) has been very successful at eliminating false-positives.10,11 However,GC/MS requires extraction, repeated calibration, and long analysis times (typically 20 to 60 minutes),11 making it laborintensive and less than desirable for field use. More rapid analysis of agents in the solid, liquid and gas phase has beendemonstrated by vibrational spectroscopy.12-15 Hoffland et al.12 reported infrared absorbance spectra and absolute Ramancross sections for several chemical agents, while Christesen measured Raman cross sections for sarin, tabun, mustard gas,and VX (ethyl S-2-diisopropylamino ethyl methylphosphonothioate).16 Again, however these techniques also havelimitations. Raman spectroscopy is simply not a very sensitive technique, and detection limits are typically 0.1% (1000ppm). While infrared spectroscopy would have limited value in analyzing poisoned water, since the very strong infrared* To whom correspondence should be addressed, email:farqu@real-time-analyzers.comSPIE-4378-2001 21
  3. 3. absorption of water would obscure most other chemicals present. Nevertheless, efforts to overcome these limitations havebeen demonstrated. Braue and Pannella13 quantified the G-series nerve agents (tabun, sarin, and soman) in terms of infraredattenuated total reflectance using a circle-cell. And Alak and Vo-Dinh demonstrated the possibility of surface-enhancedRaman spectroscopy (SERS) to identify CWAs by measuring several organophosphonates that simulate the nerve agents.17However, quantitative measurements have not been demonstrated for the SER-active material used (silver coated on aluminaparticles) or other SER-active media.18Recently, we developed silver-doped sol-gels to promote the SER effect.19-22 The porous silica network of the sol-gel matrixoffers a unique environment for stabilizing SER-active metal particles, and the sol-gel provides a high surface area thateffectively increases the number of molecules observed within the Raman scattering volume. The silver-doped sol-gels havebeen coated on the internal walls of standard glass vials to allow rapid SER analysis of small sample quantities (< 0.1 mL)without preparation. We have used p-aminobenzoic acid (PABA) as a test chemical to demonstrate surface enhancementsgreater than 107, reversible measurements in a flowing system, reproducible measurements from vial-to-vial and batch-to-batch, and measurements in multiple solvents, including water.19-22 Here we present preliminary measurements of chemicalagent simulants, in both the liquid and gas phases, as well as chemical agent hydrolysis products using our SER-active vials. 2. EXPERIMENTALThe chemical agent simulants employed were obtained at their purest commercially available grade from Aldrich(Milwaukee, WI) and were dissolved in water or methanol for analysis. All chemicals used to prepare the silver-doped sol-gels were spectroscopic grade and also purchased from Aldrich. The sol-gel vials were coated in a manner similar to thatpreviously reported by adding ammonium hydroxide to a solution of silver nitrate, tetramethyl orthosilicate, andmethanol.22 After mixing, 0.2 mL of the sol-gel solution was transferred into a glass vial (2 mL), dried and heated. Theincorporated silver ions were then reduced using dilute sodium borohydride. The vials were washed and dried prior to theaddition of a sample solution. The patent pending SER-active vials are commercially available from Real-Time Analyzers(Simple SERS Sample Vials, RTA, East Hartford, CT).Dimethyl metylphosphonate (DMMP), pinacolyl methylphosphonate (PMP) and methylphosphonic acid (MPA) wereprepared in aqueous solution, while 2-chloroethyl ethyl sulfide (CEES) was prepared in methanol at 1 mM for SERSmeasurements. Neat samples were employed for normal Raman measurements. All samples were prepared in a chemicalhood and transferred into plain or SER-active vials for analysis. Special precaution was followed for CEES, since it is asevere blistering agent.23 Once prepared, the vial was placed into the sample compartment of a Raman spectrometer foranalysis. A prototype Fourier transform Raman spectrometer (RTA) was used for these measurements.24 The systemconsisted of a Nd:YAG laser (Brimrose) for excitation at 1064 nm, an interferometer built by On-Line Technologies (OLT,East Hartford, CT) for frequency separation, an uncooled InGaAs detector for signal detection (RTA), and an Intel 400 MHzPentium II based laptop computer (Dell, Round Rock, TX) for interferometric control, data acquisition (OLT), and analysis(LabVIEW by National Instruments, Austin, TX). Additional components included a Notch filter (Kaiser, Ann Arbor, MI)and interferometer entrance and exit optics (Edmund Scientific, Barrington, NJ). Fiber optics were used to deliver theexcitation beam to the sample and the scattered radiation to the interferometer (1 meter lengths of 200 and 365 micron corediameter, respectively, Spectran, Avon, CT). A second Notch filter (Kaiser) was used as a beam splitter to direct theexcitation beam along the same axis as the collected radiation. A microscope object (20x0.4, Newport, Irvine, CA) was usedto focus the beam into the sample and to collect the scattered radiation back along the same axis. In this co-axialbackscattering arrangement, the excitation beam was passed through the outside of a glass vial and focused onto the silver-doped sol-gel film (0.1 mm thickness) containing the sample. 3. RESULTS AND DISCUSSIONAs a prelude to chemical agent measurements in water, we evaluated the quantitative performance of the SER-active vials bymeasuring PABA over the concentration range from 10-7 M to 10-2 M. Figure 1 shows the spectra for 7, 35, and 70micromolar concentrations, while Figure 2 shows a plot of the 1450 cm-1 band intensity as a function of concentration. TheSER response is linear over nearly three orders of magnitude to just over 10-4M, at which point the band intensity suggeststhat the silver surface is becoming saturated.SPIE-4378-2001 22
  4. 4. 2 10 1 10 A 0 10 B 10 -1 C -2 10500 1000 1500 2000 -7 -6 -5 -4 -3 -2 -1 10 10 10 10 10 10 10 Wavenumbers (∆cm-1) Concentration (M) Figure 1. SER spectra of A) 70, B) 35, and C) 7 micromolar Figure 2. SER spectral intensity for p-aminobenzoic acid p-amino benzoic acid in water. Conditions: 80 mW of 1064 as a function of concentration using RTA SER-active vials. nm laser excitation, 100 averaged scans (1.5 min) at 8 cm-1 resolution.In an effort to demonstrate the broad capabilities of the SER-active vials to measure chemical agents, spectra of a nerve agentsimulant: dimethyl methylphosphonate, a mustard gas simulant: 2-chloroethyl ethyl sulfide, and hydrolysis products:pinacolyl methylphosphonate and methylphosphonic acid were collected. DMMP is widely used by the U.S. Army as achemical warfare simulant because its chemical structure, volatility, and water solubility are similar to those of nerveagents.25 DMMP is completely miscible and stable in water at room temperature.26 Figure 3 compares the SER spectrum tothe normal Raman spectrum of DMMP. A number of the normal Raman bands are SER-active, such as the P-C stretchingmode which shifts from 715 to 735 cm-1, and the C-H stretching modes at 2855, 2930, 2960, and 3000 cm-1, which shiftslightly. Surprisingly, the P=O stretching mode at 1250 cm-1 virtually disappears. However, the most dramatic change is theappearance of an intense triplet in the SER spectrum near 1000 cm-1. The bands at 1000 cm-1, 1030 cm-1, and 1075 cm-1likely involved the P-O-C bond. This is supported by the nearly identical triplets observed for the SER spectra of fonofosand fonofoxon.17,19 It is also worth noting that a band appears at 425 cm-1 in the SER spectrum, that may be unique toDMMP and useful for identification. The enhancement factor is estimated at 120,000 based on the normal Raman and SERP-C band intensity, taking into account the difference in sample concentrations and spectral acquisition conditions. Adetection limit based on a signal-to-noise ratio of 3 can be estimated at 1.6 ppm. O Cl-CH2-CH2-S-CH2-CH3 = A CH3O-P-OCH3 _ CH3 A B B Wavenumbers (∆cm-1) Wavenumbers (∆cm-1)Figure 3. A) SER and B) normal Raman spectra of Figure 4. A) SER and B) normal Raman spectra of 2-dimethyl methylphosphonate. Conditions: SERS as in chloroethyl ethyl sulfide. Conditions as in Figure 3.Figure 1, normal Raman, 500 mW and 200 scans.SPIE-4378-2001 23
  5. 5. 2-Chloroethyl ethyl sulfide, a blister agent simulant, has a chemical structure similar to the mustard gas (Cl-CH2-CH2-S-CH2-CH2-Cl), with only one terminal chlorine. Due to its low solubility in water, CEES was dissolved in methanol for the SERmeasurement. Again the prominent Raman modes are SER-active and even maintain relative intensity (Figure 4). Theprimary difference is that the SER bands appear to broaden, such that the triplet near 700 cm-1 becomes a doublet and theshoulders at 2875 and 2970 cm-1 become less defined. Again, the latter bands are assigned to C-H stretching modes. Asingle band at 700 cm-1, which is attributed to the C-S-C asymmetric stretch, dominates the reported infrared spectrum ofmustard gas.12 A corresponding symmetric stretch is reported at 705 cm-1 in the Raman spectrum of mustard gas.27 Here acorresponding symmetric stretch appears, but as a doublet at 700 and 755 cm-1, presumably due to the loss in symmetry forCEES. The band at 655cm-1 can also be confidently assigned to a C-Cl stretch. The SER spectral bands at 620 and 730 cm-1are probably due to the same modes, i.e. C-Cl and C-S-C stretches, respectively. The enhancement factor for CEES wassomewhat less than DMMP at approximately 62,000, as is the estimated detection limit of 2.2 ppm.The ability to rapidly detect trace quantities of chemical agents in the gas phase would be invaluable as an early warningsystem. Although the Raman scattering cross-sections for the nerve agents suggest that remote detection by Raman-basedLIDAR is unlikely,16 a SER-based system for perimeter monitoring could prove successful. As a preliminary measurement,we prepared a 10% by volume solution of CEES in methanol, exposed a SER-active vial to the equilibrium vapor phase in asealed jar, and monitored the SER spectrum as a function of time. Initially, the vial was removed through a transfer chamberevery hour to record the SER spectrum. After ten hours, spectra were recorded only every ten hours. As illustrated by Figure5, the sol-gel performed as a dosimeter, in that the spectra increased as a function of exposure time. The most intense SERbands at 620 and 2930 cm-1 are discernable in the first few hours. The spectrum after 40 hours is nearly identical to thesolution phase spectrum, except for a diminished intensity of the 730 cm-1 band. This may be due to methanol solvationeffects or surface-orientation effects. Based on the relative concentrations of methanol and CEES and their partial pressures,we estimate the equilibrium concentration of CEES to between 1 and 2 micromolar. Although not shown, this concentrationcould be detected in one hour. O = A HO-P-OH _ CH3 O = CH3 _ B HO-P-O-CH-C-CH3 _ _ CH3 CH3 CH3_ Wavenumbers (∆cm-1) Wavenumbers (∆cm-1)Figure 5. SER spectra of 2-chloroethyl ethyl sulfide Figure 6. SER spectra of A) methyl phosphonic acid andvapor as a function of time (10 hour increments to top, B) pinacolyl methylphosphanate (note unique band atwhich is 40 hours). Bottom trace is a blank. Spectral 546 cm-1). Spectral conditions as in Figure 1.conditions as in Figure 1.As previously stated, the analysis of chemical agents in water is important in identifying poisoned water. It is also importantto decommissioning activities, in which agents are destroyed by hydrolysis (acid or base). Furthermore, any analyticaltechnique used must be capable of distinguishing between parent CWA and hydrolysis products to assess safety oreffectiveness of decommissioning. For example, soman has a hydrolysis half-life of ~2.3 hours at ambient temperatures andneutral pH,28 and forms hydrofluoric acid (somewhat toxic) and pinacolyl methylphosphonate (relatively non-toxic).29,30PMP further hydrolyzes to form methyl phosphonic acid and 3,3-dimethyl-2-butanol (both non-toxic). The structuralsimilarities between soman, PMP and MPA are expected to produce similar Raman, as well as SER spectra. Figure 6compares PMP and MPA, but not the highly toxic parent CWA soman. As with DMMP, the P-C stretch, the P-O-C mode,and C-H stretches are readily apparent. Yet it is worth noting that the band positions are reasonably different. The formertwo bands appear at 764 and 1042 cm-1 for MPA, while they are at 788 and 1032 cm-1 for PMP. More importantly, a uniqueband at 546 cm-1, as yet unassigned, appears in the PMP spectrum.SPIE-4378-2001 24
  6. 6. 4. CONCLUSIONSWe have successfully measured the SER spectra of chemical agent simulants: dimethyl metylphosphonate and 2-chloroethylethyl sulfide, and chemical agent hydrolysis products: pinacolyl methylphosphonate and methylphosphonic acid, using silver-doped sol-gel coated sample vials. Measurements were obtained in both aqueous and gas phase. The P-C stretching modewas SER-active for all four chemicals, allowing identification by class. Within this group, each chemical contained at leastone unique spectral band that could be used for identification (Table 1). Furthermore, these bands do not appear to coincidewith SER spectra reported for organophosphorus pesticides, the most likely source of false-positives. Although surfaceenhancement factors appear to be an order of magnitude better than those previously presented in the literature for similarchemicals,17 measurement sensitivity needs to be improved by 1 to 2 orders of magnitude to provide adequate warning ofchemical agent use. Current research efforts to increase surface-enhancement, optical collection efficiency, and instrumentdesign are being pursued to achieve the required sensitivity. Table 1. Enhancement factors, detection limits and unique SER bands fro chemicals studied. Agent Simulant Enhancement Detection limit Unique bands (cm-1) Dimethyl methylphosphonate 120,000 90 µM (1.6 ppm) 425 2-Chloroethyl ethyl sulfide 62,000 60 µM (2.2 ppm) 620 Methylphosphonic acid 110,000 3 µM (60 ppb) 764, 1042 Pinacolyl methylphosphonate 150,000 70 µM (1.4 ppm) 546, 788, 1032 5. ACKNOWLEDGEMENTSThe authors would like to thank Drs. Janet Jensen and Steven Christesen of Aberdeen Proving Ground for encouraging thiswork. They would also like to thank Advanced Fuel Research for making their laboratory facilities available. 6. REFERENCES1 “The Chemical Weapons Convention – A Guided Tour, the Organization for the Prohibition of Chemical Weapons” at http://www.opcw.nl/guide.htm.2 Robinson, J.P. and J. Goldblat, "Chemical Warfare In The Iraq-Iran War" Stockholm International Peace Research Institute Fact Sheet, at http://projects.sipri.se/cbw/research/factsheet-1984.html (1984)3 “Chemistry of GB (Sarin)” at http://www.mitretek.org/mission/envene/chemical/agents/sarin.html.4 Tu, Anthony, “Overview of Sarin Terrorist Incidents in Japan in 1994 and 1995”, 6th CBW Protection Symposium, Stockholm, Sweden, 10-15 May 1998.5 Staff Reporter, “Going out with a bang”, Newsweek, June 28, 1999.6 See UNSCOM reports in http://www.un.org/depts/unscom (1999).7 Treven, T., Saddam’s Secrets, Harper Collins (1999)8 “Decaying Sarin-filled Rockets Spark Fears”, Jane’s Defense Weekly, 25(20),3 (1996).9 “The Chemical Weapons Convention Redefines Analytical Challenge”, Analytical Chemistry News & Features, June 1 397A (1998).10 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)11 Black, R.M., Clarke, R.J., Read, R.W., and Reid, M.T., “Application of gas chromatography-mass spectrometry and gas chromatography-tandem mass spectrometry to the analysis of chemical warfare samples, found to contain residues of the nerve agent sarin, sulphur mustard and their degradation products”, J. Chromatography, 662, 301-321 (1994)12 Hoffland, L.D., Piffath, R.J., Bouck, J.B.,”Spectral signatures of chemical agents and simulants”, Optical Engineering, 24, 982-984, (1985)13 Braue, E.H.J., Pannella, M.G.,”CIRCLE CELL FT-IR Analysis of Chemical Warfare Agents in Aqueous Solutions”, Applied Spectroscopy, 44, 1513-1520, (1990)SPIE-4378-2001 25
  7. 7. 14 Tseng, C.-H., Mann C.K., and Vickers T.J., “Determination of Organics on Metal Surfaces by Raman Spectroscopy”, Applied Spectroscopy, 47, 1767-1771 (1993)15 Staff Reporter, “US Army Tests Lidar to Detect Biological Toxins”, Photonic Spectra, pg. 50, December 1998.16 Christesen, S.D., "Raman cross sections of chemical agents and simulants", Applied Spectroscopy, 42, 318-321 (1988)17 Alak, A.M. and Vo-Dinh, T., “SERS of Organophosphorous Chemical Agents”, Analytical Chemistry, 59, 2149-2153 (1987)18 Norrod, K.L., Sudnik, L.M., Rousell, D., and Rowlen, K.L., “Quantitative Comparison of Five SERS Substrates: Sensitivity and Detection Limit”, Applied Spectroscopy, 51, 994-1001 (1997).19 Lee, Y. and Farquharson, S., “SERS Sample Vials Based on Sol-Gel Process for Trace Pesticide Analysis”, SPIE, 4206, 140-146 (2000).20 Farquharson, S. and Lee, Y., “Trace Drug Analysis by Surface-Enhanced Raman Spectroscopy”, SPIE, 4200-16 (2000).21 Lee, Y., Farquharson, S., and Rainey, P. M., "Surface-Enhanced Raman Sensor for Trace Chemical Detection in Water", SPIE, 3857, 76-84 (1999).22 Lee, Y, Farquharson, S., Kwong, H., and Shahriari, M., “Surface-Enhanced Raman Sensor for Surface-Enhanced Raman Spectroscopy”, SPIE, 3537, 252-260 (1998).23 see Material Safety Data Sheets for details.24 Farquharson, S., Smith, W., Carangelo, R. C., and Brouillette, C., “Industrial Raman: Providing Easy, Immediate, Cost Effective Chemical Analysis Anywhere”, SPIE, 3859, 14-23 (1999)25 Bennett, S., Bane, J., Benford, P., and Pratt, R., “Environmental Hazards of Chemical Agent Simulants”, Aberdeen Proving Ground, Maryland: Chemical Research and Development Center, CRDC-TR-84055 (1984).26 Mabey, W. and Mill, T., Critical Review of Hydrolysis of Organic Compounds in Water under Environmental Conditions. Journal of Physics and Chemistry Reference Data, 7(2): 383-414 (1978).27 Christesen, S., MacIver, B., Procell, L, Sorrick, D., Carabba, M, and Bello, J., “ Noninstrusive Analysis of Chemical Agent Identification Sets Using a Portable Fiber-Optic Raman Spectrometer”, Applied Spectroscopy, 53, 850-855 (1999).28 Meylan, W.M. and Howard, P.H., J. Pharm. Sci., 84, 83-92 (1995)29 Jenkins, A., Uy, O. and Murray, G., “Polymer-Based Lanthanide Luminescent Sensor for Detection of Hydrolysis Product of the Nerve Agent Soman in Water”, Analytical Chemistry, 71, 373-378 (1999).30 Nassar, A., Lucas, S., and Hoffland, L., “Determination of Chemical Warfare Agent Degradation Products at Low-Part- per-Billion Levels in Aqueous Samples and Sub-Part-per-Million Levels in Soils Using Capillary Electrophoresis”, Analytical Chemistry, 71, 1285-1292 (1999).SPIE-4378-2001 26
  8. 8. Appendix B Detection of bioagent signatures: A comparison of electrolytic and metal- doped sol-gel surface-enhanced Raman media Stuart Farquharson,* Wayne Smith, and Yuan Lee Real-Time Analyzers, 87 Church Street, East Hartford, CT 06108 Susan Elliott and Jay F. Sperry University of Rhode Island, 45 Lower College Rd, Kingston, RI 02881 ABSTRACTSince September 11, 2001, the threat of terrorist attacks and biological warfare within U.S. borders has become a soberingreality. In an effort to aid military personnel and the public at large, we have been investigating the utility of surface-enhanced Raman spectroscopy (SERS) to provide rapid identification of chemical agents directly, and biological agentsthrough their chemical signatures. This approach is based on the ability of Raman spectroscopy to identify molecularstructure through the abundant vibration information provided in spectra and the ability of SERS to detect extremely lowconcentrations (e.g. part-per-billion) through the enhancement of Raman scattering by six orders of magnitude or more.Towards the goal of developing a portable analyzer, we have been studying the ability of two SER media to obtaincontinuous (i.e., reversible) and quantitative (i.e., reproducible) measurements. Here we compare measurements of nucleicacid-bases, adenosine monophosphate, and ribonucleic acid extracted from Escherichia coli, Bacillus subtilis andStaphylococcus aureus obtained by electrolytic SERS and metal-doped sol-gel SERS. The capabilities of these SER mediaare summarized in terms of rapid detection of B. anthracis and dipicolinic acid.Keywords: bioagent detection, SERS, RNA analysis, bacterial analysis, Raman spectroscopy 1. INTRODUCTIONThe recent distribution of anthrax through the U.S. postal system and the subsequent infection and death of several postal andnational media employees, amplifies the need for methods to rapidly detect and identify this and other chemical andbiological warfare agents (BWA). The primary methods currently used, immunoassays for screening and nucleic acid (NA)sequencing for positive identification of BWAs (bacteria, protozoa and viruses), have serious limitations.1,2,3 Immunoassaymethods employ competitive binding of the bioagent (as an antigen) and its labeled (e.g. fluorescence) conjugate for a limitednumber of antibodies. Although this analysis method is fast and semi-quantitative, other chemicals may compete for theantibodies, interfere with the enzymatic reaction or interfere with the measurement (e.g. it fluoresces) resulting in a highnumber of false positive responses.1 Furthermore, the antibodies denature due to moisture and heat, limiting shelf life, andrequire sterile, often refrigerated storage. Positive identification of a BWA can be accomplished by sequencingdeoxyribonucleic acid or ribonucleic acid (DNA and RNA).2,3 This requires enumeration of a nucleic acid sequence throughpolymerase chain reactions (PCR) or multiplication of the microorganism through culture growth to provide sufficientquantities of DNA or RNA for analysis. Unfortunately, PCR and culture growth require from several hours to several days.2,3Consequently, a wide variety of technologies have been investigated for rapid identification of BWAs. The Department ofDefense is actively monitoring 200 such technologies.4 This includes traditional methods, such as gas chromatographicseparation coupled with ion mobility spectrometry detection,5 to exotic methods based on nature, such as monitoring toxininduced color changes in fish scales.6 Although all of these techniques have achieved varying degrees of success, none areyet capable of detecting and identifying BWAs in 10 minutes or less. Towards this goal we have been investigating theability of SERS to detect sub-nanogram quantities of DNA or RNA (eliminating enumeration), determine relative NA baseconcentrations, and identify BWA taxonomy.* To whom correspondences should be addresses, e-mail:farqu@real-time-analyzers.com, www.real-time-analyzers.comSPIE 2001-4575 62
  9. 9. Raman spectroscopy has a rich history of investigating biochemical and biological processes.7 Some of the earliest laser-Raman studies demonstrated that the five NA bases, adenine (A), cytosine (C), guanine (G), thymine (T, in DNA) and uracil (U, in RNA), yielded distinct spectra with several bands suitable for identification and quantification.8 Furthermore, thesestudies included exceptional spectra of both DNA and RNA, for which the NA bases, as well as several phosphate bandswere easily identified.9 However, since the Raman effect is very inefficient (very low conversion of incident radiation toinelastically scattered Raman radiation), these samples had to be highly concentrated.Fortunately, two phenomena exist that can increase the generation of Raman photons by six orders of magnitude or more,known as the resonance Raman and surface-enhanced Raman effects.10,11 Resonance Raman scattering occurs when the laserexcitation wavelength is in resonance with an electronic transition of a molecule (within the absorption envelope).10Excitation at ultraviolet wavelengths has been used to obtain resonance Raman spectra of amino acids and nucleic acids inwhole bacteria.12,13 For example, excitation at 242 nm has been used to maximize the nucleic acid spectral band intensities,and minimize the amino acids band intensities. A peak at 1530 cm-1 was found to be proportional to the amount of the NAbase cytosine, while a peak at 1485 cm-1 was proportional to the combined amount of the NA bases adenine and guanine.This quantitative behavior has been used to define an A+T/G+C base-pair ratio and provide a level of bacterial identificationas taxonomic markers.13In recent years SERS has also been used to analyze bacterial cell components,14 including amino acids,15 lipids,16 nucleicacids,15,17,18 and the adenine derivatives.19,20,21 SERS has proven to be one of the most sensitive methods for trace chemicalanalysis through the detection of single molecules,22,23 including DNA (dye labeled 17-mer).24 Since its discovery in 1974,25the mechanism responsible for the large increase in scattering efficiency has been the subject of considerable research.26,27Briefly, incident laser photons couple to free conducting electrons within a metal, which confined by the particle surface,collectively cause the electron cloud to resonate.26,28 These surface plasmons are known as the physical component of theSER effect. These surface plasmons can transfer energy to the molecular vibrational modes of molecules throughinteractions with the molecular electron orbitals.26,29 This interaction is known as the chemical component of the SER effect.This perturbation of the molecular polarizability generates surface-enhanced Raman photons.26A number of methods have been developed to produce surfaces or solutions containing one of these metals with optimumroughness or diameter to promote SERS.30 These methods include preparation of activated electrodes in electrolyticcells, 11,31 activated silver and gold colloid reagents,32 and metal coated substrates.33,34,35 Selecting a SER-active medium forchemical and biological agent detection requires consideration of the method of deployment, and hence the method ofsampling. Chemical aerosols or airborne bacteria will require a collection device to concentrate and continuously present thesample to the SERS medium. Poisoned water supplies will also require a flow through device for continuous monitoring, ora grab-sample device for periodic analysis. And contaminated surfaces will require a grab-sample extractive device. ASERS-based device used for continuous monitoring (air or water) must be reversible and reproducible if quantitativemeasurements are desired, while a SERS-based device used for periodic sampling (water or surfaces) must be reproducible.Both reversible and reproducible measurements have been performed using electrolytic SERS (E-SERS).36 But this requiresa three-electrode sample cell and an electrolyte of known concentration to perform the necessary oxidation-reduction cycles(ORCs) to re-activate the electrode surface with new, uncontaminated sites from one measurement to the next. Colloids areseverely limited, in that continuous measurements would require a continuous supply of colloids. For periodicmeasurements, vials of colloids, one per measurement, could be used. However, aggregate size and consequently SERintensity change with sample conditions (especially pH), and quantitative, reproducible measurements are unlikely.Substrates appear to have the greatest potential, and designs range from silver evaporated on titania particles34 to periodicgold pyramids evaporated between polystyrene beads.35 Most substrates require concentrating the sample on the surfacethrough drying to obtain the largest signal enhancements, in effect making the measurements irreproducible and irreversible.However, successful measurements using flow systems have been obtained with glass posts, but manufacturing costs appearprohibitive.In an effort to overcome these limitations, we have developed metal-doped sol-gels to provide SERS measurements that arereproducible, reversible, and quantitative, and yet not restricted to specific environments, such as electrolytes, solvents, orevaporated surfaces.37,38 The porous silica network of the sol-gel offers a unique environment for stabilizing SER activemetal particles, and the high surface area increases the interaction between the analyte and metal particles. The sol-gel can becoated on the end of fiber optics or on the internal walls of a glass flow tube for continuous measurements or standard glasssample vials for periodic measurements. Previously we measured 100 mg/L methylphosphonic acid (the primary hydrolysisproduct of nerve agents) in water with an estimated detection limit of 0.5 mg/L (100 parts-per-billion). We have alsoSPIE 2001-4575 63
  10. 10. demonstrated reversible and reproducible measurements of p-aminobenzoic acid (PABA) in a flow through system. Here weinvestigate the ability of the sol-gel SERS (SG-SERS) to measure the NA bases, adenosine monophosphate, and RNAextracted from E. coli, B. subtilis and S. aureus. The measurements are compared to those obtained by E-SERS. 2. EXPERIMENTALThe inorganic chemicals and solvents used to prepare samples were spectroscopic grade and purchased from Aldrich(Milwaukee, WI), Fisher (Pittsburgh, PA) or Pfaltz & Bauer (Waterbury, CT). The nucleic acid bases and dipicolinic acidwere purchased from Sigma (St. Louis, MO). Normal Raman samples were measured to establish enhancement factors. Ineach case 1cm3 of sample was placed into a 1x1 cm glass cuvette weighed and measured. Unpacked densities were typically6-7 g/cm3. For all SER measurements, including RNA, samples were prepared as ~0.1mg/mL (see Figure captions for exactconcentrations) in 0.1M KCl and buffered to a pH of 9.2 with Na2B4O7•H2O. Adenine pH dependence measurements usedpH buffer solutions at 4 (potassium acid phthalate), 6.9 (potassium phosphate monobasic/sodium phosphate dibasic), 9.2,(Na2B4O7•H2O) and 10.4 (tris-hydroxymethyl amino methane). Escherichia coli, Bacillus subtilis and Staphylococcus aureuscultures (250ml per 1000mL Erlenmeyer flask) were grown overnight in a Trypticase soy broth (TSB) medium containing1% glucose in a shaking water bath at 37 oC. The bacteria were harvested by centrifugation for 10 minutes at 8,000 rpm in aGSA rotor at 5°C, then washed once in 0.85% saline. The gram-positive bacteria were concentrated to 20 ml and passedthrough a French pressure cell twice at 15,000 psi to break open the cells. RNA was extracted according to Protocol 4.41,39to ensure pristine samples for initial measurements. Since this method takes approximately 4 hours, a streamlined methodwas developed. For vegetative bacteria, the specimen was boiled for 30 sec in 1 ml of distilled water to lyse the cells andrelease the RNA. For bacterial spores, the specimen was first incubated in 1 ml of saline solution containing 0.2 mglysozyme and phosphate-buffered to pH of 6.24 for 1 hr at 37 oC. This solution was then boiled for 2-3 minutes in 4Xloading buffer to release the RNA. For both specimens, RNA STAT-60TM was added to the supernatant, which wascentrifuged at 12,000 g for 5 minutes to precipitate the ~15% water-soluble proteins. This procedure allowed extractingRNA for SER analysis in ~ 10 minutes. Electrophoresis shows high purity, while the existence of chemicals that couldinterfere with the SER measurements is still under investigation.The electrolytic sample cell has been described previously.36 Briefly, a three electrode design is incorporated into a Plexiglasstructure containing a well for the reference electrode (a saturated calomel electrode, Cole Parmer, Vernon Hills, IL) and a5mL sample well containing the silver working electrode and platinum wire counter electrode (0.5 mm wire, Alfa, Ward Hill,MA). A channel connecting the two wells contained a 2 mm diameter semi-porous membrane (10-20 micron pore, AceGlass). The silver electrode was made from a 3 mm length of 2 mm diameter silver wire (Alfa) soldered to a copper wirelead encased in a 4 mm diameter Pyrex tube. A cap containing the silver electrode, platinum wire, and nitrogen purge andvent lines fixed the silver electrode surface 1 mm from a 1 mm thick glass plate attached to the bottom of the sample well.The potentiostat used to control the three electrodes was built in-house and has been described in detail elsewhere.36 Amultifuntional analog, digital, and timing input/output interface card (DAQCard-1200, National Instruments) is used to bothdrive the electrolytic cell as well as read the current generated in the cell. A LabVIEW software program is used set theoxidation potential, reduction potential, SER measurement potential, hold times, and sweep rates. The amount of chargepassed was plotted as a cyclic voltammogram. For all spectra presented, five oxidation-reduction cycles (ORCs), steppingfrom -0.3 VSCE to 0.3 VSCE and back to -3 VSCE at 50 mV/sec were used.The SG-SER measurements were accomplished by simply placing the identical samples prepared above into Simple SERSSample VialsTM (RTA). These 2-mL, glass vials are internally coated with ~ 0.1 micron thick silver-doped sol-gel.A prototype Fourier transform Raman spectrometer (RTA) was used for these measurements.40 The system consisted of aNd:YAG laser (Brimrose or Spectra Physics) for excitation at 1064 nm, an interferometer built by On-Line Technologies(OLT, East Hartford, CT) for frequency separation, an uncooled InGaAs detector for signal detection (RTA), and an Intel 400MHz Pentium II based laptop computer (Dell, Round Rock, TX) for interferometric control, data acquisition (OLT), andanalysis (LabVIEW by National Instruments, Austin, TX). Additional components included a Notch filter (Kaiser, AnnArbor, MI) and interferometer entrance and exit optics (Edmund Scientific, Barrington, NJ). Fiber optics were used todeliver the excitation beam to the sample and the scattered radiation to the interferometer (2 meter lengths of 200 and 365micron core diameter, respectively, Spectran, Avon, CT). A second Notch filter (Kaiser) was used as a beam splitter to directthe excitation beam along the same axis as the collected radiation. A microscope object (20x magnification, 0.4 numericaperture, Newport, Irvine, CA) was used to focus the beam into the sample and to collect the scattered radiation back alongthe same axis. In this co-axial backscattering arrangement, the excitation beam passed through the glass plate onto the silverSPIE 2001-4575 64
  11. 11. electrode surface for E-SERS, through the vial glass wall and into the silver-doped sol-gel film for SG-SERS, or through theglass wall of the cuvette and into the solid sample for normal Raman spectroscopy. All E-SERS and normal Raman spectrawere obtained with 750 mW of laser power at the sample, while all SG-SERS spectra were obtained with 75 mW of laserpower at the system. Incident powers above 200 mW in some cases degraded the sol-gel. 3. RESULTS AND DISCUSSIONThe generation of surface-enhanced Raman scattering at electrode surfaces has been extensively researched, and the optimumsample conditions are well developed.27,29 Several researches incorporated electrodes into flowing systems and demonstratedthat continuous monitoring of chemicals is possible.18 These successes suggested investigated the capability of E-SERS tomeasure the NA bases and RNA. The E-SERS measurements also provided a benchmark to compare and evaluate SG-SERSmeasurements. The molecular structure of adenine (as well as the other base pairs), which includes an aromatic nitrogen-containing heterocycle, is ideally suited to interact with the surface plasmons and contribute substantially to the chemicalcomponent of the SER effect.11,19 Even with excitation at 1064 nm, a 3-minute scan of 1.8x10-5M adenine yields high signal-to-noise (S/N) E-SER spectra and all of the bands are revealed with clarity (Figure 1, Table 1). Spectra of similar qualitywere obtained by SG-SERS and the principal spectral bands are easily observed. The identical 1.8x10-5M adenine samplewas measured in the same 3-minute time frame, but with 1/10th the laser power. The lower power appears to reduce the S/N. 725 A A 735 pH 10 B B SG-SERS C 735 C D pH 4 Wavenumbers (∆cm-1) Wavenumbers (∆cm-1)Figure 1. A) Normal Raman spectrum of pure adenine Figure 2. A) and C) E-SERS and B) and D) SG-SERS ofpowder, B) E-SERS and C) SG-SERS of 1.8x10-5M adenine at A) and B) pH 10.4 and C) and D) pH 4.0. Noteadenine at pH 9.2. All spectra 8 cm-1 resolution, 200 scans consistent appearance of bands at 1270 and 1375 cm-1 as(3 min), and 1064 nm excitation. A) and B): 750 mW, C) the pH is changed to 10 for both SER media. E-SERS75 mW. B) measurement potential of 1.1VSCE. used 750 mW, SG-SERS used 75 mW of 1064 nm excitation.The amount of adenine responsible for the SER spectra, as well as enhancement factors for the two SER media can bedetermined. The molecules producing the E-SERS spectrum are those on the electrode surface within the illumination areaof the laser. (The solution concentration only determines the number of molecules available to adsorb to the electrodesurface.) For the current experiments the laser illuminates an area of 2.8x10-7m2, or 5.6 x10-7m2 if we assume the ORCsincrease the surface area by a factor of two. Furthermore, if we assume monolayer coverage on the electrode and each 3x5angstrom molecule (lying flat) occupies 1.5x10-19m2, then there are ca. 4x1012 molecules contributing to the Ramanscattering. This is ca. twice the number of molecules measured at electrode surfaces using either differential capacitance-potential curve measurements or rapid linear sweep voltammetry (e.g. 3x1018 molecules/m2 for pyridine and pyrazine).29Thus the adenine spectrum in Figure 1 is due to 8.7x10-10g (6x10-12 moles)! A detection limit defined as a S/N of 3 can alsobe calculated. The S/N for a 3-minute scan is 844 for the 735 cm-1 band, suggesting a mass detection limit of 3x10-12g(2x10-14 moles). This is consistent with previous estimates for adenine by others of 2.5 x10-14 moles.15,30 However, sub-monolayer concentrations must be measured to verify this. The root-mean-squared (RMS) noise is measured between 4400-4600 cm-1. Since noise is distributed evenly throughout the spectrum when transformed, this region does not have anySPIE 2001-4575 65
  12. 12. contributions from signals or baseline offsets. The measurement error is given as S±RMS, and for adenine this equals 2.34%.The number of molecules contributing to the SG-SERS are those on the silver particles that are embedded in the sol-gel. Thetotal silver surface area can be determined from the average particle size (40 nm diameter), concentration (0.73% by weight,based on molar conc. and measured sol-gel density), and the scattering volume (a cylinder defined by the laser area:2.8x10-7m2 and sol-gel thickness:10-4m). The 6.1x109 silver particles in this volume have a collective area of 3.1x10-5m2.However, it may reasonably be assumed that at least half of the silver surface is in contact with the sol-gel matrix andunavailable for analyte interaction. Then approximately 1.0x1014 molecules or 2.2x10-8g of adenine contribute to the SG-SER spectrum. The slightly lower S/N of 207 suggests a mass detection limit of 3.2x10-10g.Determination of the enhancement factors for the two SER media requires estimating the number of adenine moleculescontributing to the normal Raman spectrum. Here a cylindrical scattering volume is assumed, again based on the laser area(2.8x10-7m2) and the penetration depth (1x10-3 m).41 The density of the sample was measured at 0.64 g/cm3, indicating that1.8x10-4g (1.3x10-6 moles) of adenine produced the normal Raman signal. The enhancement factor, EF, is defined by thefollowing equation: EF = (ISERS/INR)•(MNR/MSERS) •(PNR/PSERS) •(TNR/TSERS)1/2where I is the spectral band intensity (here 735 cm-1), M is the sample mass, P is the incident laser power, and T is themeasurement time (or number of scans). For the E-SERS measurement the enhancement factor is 2.2x105 (0.178/0.184) •(1.8x10-4/8.7x10-10)), while the SG-SERS enhancement factor is 1.0x105 (0.16/0.184) •(1.8x10-4/2.2x10-8) •(750/75) •(3/1.5)1/2).The lower enhancement for the SG-SERS may be real, or the available surface of the silver embedded in the sol-gel mayhave been overestimated.In addition to enhancing the Raman scattering efficiency to an extent similar to E-SERS, the SG-SER medium also yields anidentical shift of the adenine ring-breathing mode from 725 cm-1 in the normal Raman to 735 cm-1. Furthermore, in thecourse of optimizing the E-SERS sample conditions, it was found that pH influenced the adenine interaction with the silversurface (Figure 2). In particular, the relative band intensities of the pyrimidine ring skeletal vibrations at 1270 and 1375 cm-1,and the imidazol ring skeletal vibration at1335 cm-1 change. At pH 4 adenine is protonated, presumably the imidazol ring,since the band at 1335 cm-1 increases in intensity, while the pyrimidine bands are virtually absent. Conversely at pH 10, theimidazol band decreases in intensity, while the pyrimidine bands appear. It is worth noting that the ring-breathing mode at735 cm-1 changes little between pH 4 and 10, suggesting that the skeletal changes are more a function of molecule-plasmoninteractions than reorientation of the molecule on the surface. Measurements of the identical pH series of adenine samples bySG-SERS yielded virtually identical spectral changes. This suggests that the sol-gel does not influence the measurement.This is critical to reproducing measurements and performing quantitative analysis.Next, the remaining NA bases were measured by both E-SERS and SG-SERS and compared. Previously we examined theoptimum pH and electrode potentials for E-SERS measurements to determine if a common pH could be used that yieldedgood sensitivity for all the bases, and if variations in potential could be used to provide an added degree of selectivitybetween the bases. Primarily it was found that high quality spectra were obtained between pH 7 and 9.5, and that cytosineand uracil were best enhanced at potentials positive of the potential-of-zero charge (pzc, ca. 0.65VSCE for Ag), guanine andthymine near the pzc, and adenine negative of the pzc. In all cases the ring-breathing modes were the most intense and ingeneral could be used to identify the NA bases (Figure 3, Table 1). Specifically, adenine has an intense band at 735 cm-1,cytosine at 797 cm-1, guanine at 653 cm-1, thymine at 784 cm-1, and uracil at 800 cm-1. The adenine, cytosine, guanine andthymine bands are sufficiently separated that their contributions to DNA should be determinable. Although adenine andguanine contributions to RNA should also be determinable, cytosine and uracil are highly overlapped, and unfortunatelyshare the same potential dependence. Alternate, unique bands at 1183 cm-1 for cytosine and 1275 cm-1 for uracil might besuitable for calculating contributions. The SG-SER spectra of the remaining NA bases faithfully reproduced the E-SERspectra. In particular the primary identifying bands occur at virtually the same wavenumbers (see Table 1). However, thespectra for both cytosine and thymine contain an intense band at ca. 1040 cm-1. Initially this was attributed to the pH buffer,but samples prepared without either the buffer or the 0.1M KCl electrolyte yielded identical spectra containing this band. Infact, the E-SER and SG-SER spectra of thymine are virtually identical except for this band. Also, the SG-SERS of guaninecontains an intense band at 1551 cm-1 that is not observed in the E-SER spectrum. This band may be due to a moderatelyintense band at 1553cm-1 in the normal Raman spectrum that is SG-SER active. It was also found that the SG-SERS ofcytosine was considerably better than the E-SERS, while uracil showed the opposite relationship. It is also worth noting thatall of the SG-SERS were obtained with 1/10th the laser power. Most importantly, the primary ring-breathing modes in theSG-SER spectra are sufficiently intense and unique to be used in determining contributions to DNA and RNA as outlinedabove.SPIE 2001-4575 66
  13. 13. A A B B C C D D Wavenumbers (∆cm-1) Wavenumbers (∆cm-1)Figure 3. E-SERS of A) 2.1x10-3M cytosine at -0.3VSCE, Figure 4. SG-SERS of A) 2.1x10-3M cytosine, 200 scans,1000 scans, B) ~1.0x10-5M guanine at -0.6VSCE, 500 B) ~1.0x10-5M guanine, 200 scans, C) 2.3x10-3Mscans, C) 2.3x10-3M thymine at -0.6VSCE, 500 scans and thymine, 200 scans and D) 1.2x10-3M uracil, 500 scans.D) 1.2x10-3M uracil at -0.93VSCE, 500 scans. All spectra: All spectra: at pH 9.2, 75 mW 1064 nm at 8 cm-1.at pH 9.2, 750 mW 1064 nm at 8 cm-1.Table 1. Comparison of E-SER and SG-SER Spectral Band Positions for the NA Bases and Adenosine Monophosphate. Adenine Cytosine Guanine Thymine Uracil AMP E-SERS SG-SERS E-SERS SG-SERS E-SERS SG-SERS E-SERS SG-SERS E-SERS SG-SERS E-SERS SG-SERS 1647 1638 1634 1656 1655 1630 1587 1585 1510 1580* 1551* 1539* 1456 1456 1465 1460 1462 1480 1453 1459 1394 1398 1425 1431 1435 1399 1404 1392 1374 1375 1373 1383 1370 1335 1332 1311 1307 1333 1331 1353 1348 1331 1329 1265 1273 1280 1292 1278 1276 1275 1279 1271 1183 1195 1222 1232 1221 1219 1204 1205 1180* 1144 1097 1033 1029 1038 1040* 1035* 1051 1037* 1041 1035 963 963 957 1001 1000 961 944 884 819 817 859 866 735 737 797 799 784 782 800 800 727,38 742 630 630 653 664 667 684* 603 602 590 611 466 561* Bands unique to SG-SERS.The next chemical to be analyzed by both E-SERS and SG-SERS was adenosine monophosphate (AMP). The E-SERspectrum yields bands due to the adenine chemical functionality at 727, 961, 1233, 1279, 1331, 1381 and 1486 cm-1. Inaddition, phosphate bands are observed at 860, 1097, 1453, 1587, and 1705 cm-1 (Figure 5). Other researchers have notedthat the ribose component does not appear to contribute to the spectrum.19 The AMP spectrum also changes as a function ofpotential. As the electrode is swept more positive (here from -0.9 to -0.3VSCE) the phosphate bands at 860, 1097, 1453, and1587 cm-1 increase in intensity compared to the adenine bands, while a band at 1705 cm-1 appears. The adenine bands at1233, 1381 and 1486 cm-1 virtually disappear. These potential dependent spectral changes are consistent with earlier studiesthat show that phosphate is attracted to silver at potentials positive of the pzc, but repelled at potentials negative of the pzc.19SPIE 2001-4575 67
  14. 14. The SG-SER spectrum of AMP is considerably different. The adenine bands virtually disappear, except for the two primarybands, which shift to742 and 1329 cm-1. While the phosphate band at 1459 cm-1 has gained considerable intensity. Inaddition two new intense bands appear at 684 and 1539 cm-1, as well as a moderately intense band at 1180 cm-1. The SG-SER spectrum has greater similarity to the E-SER spectrum at -0.3VSCE, and suggests that the silver particles embedded in thesol-gel behave as if at a potential positive of the pzc. AMP RNA A E. coli B B. subtilis S. aureus C Wavenumbers (∆cm-1) Wavenumbers (∆cm-1)Figure 5. E-SER spectra of 0.20 mg/mL adenosine Figure 6. E-SERS of 0.1 mg/mL RNA from E. coli, 0.2monophosphate at A) -0.3 and B) -0.9VSCE, and C) SG-SER mg/mL RNA from B. subtilis and 0.2 mg/mL RNA fromspectra. Conditions: sample in 0.1M KCl buffered to pH 9.2, A) S. aureus. Conditions: 0.1M KCl, pH 9.2 -0.3VSCE, 750and B) 750 mW, C) 75 mW of 1064, 64 scans (1-min) at 8 cm-1. mW of 1064 nm, 640 scans (10 min) at 8 cm-1.RNA samples extracted from E. coli, B. subtilis and S. aureus were next examined by both E-SERS and SG-SERS. E-SERspectra of these samples yielded quality spectra in 10 minutes, in which all of the major features can be identified (Figures 6and 7). This includes guanine at 650 cm-1, adenine at 791 cm-1, cytosine and uracil combining at 790 cm-1, and phosphate at1100, 1335 (in combination with adenine and guanine), 1465 and 1570 cm-1. Surprisingly, adenine, which demonstrated thegreatest surface-enhanced Raman effect, does not dominate the ring-breathing mode portion of the spectrum. The intensitiesof the other base-pairs bands are of the same order of magnitude. This suggests that when the base-pairs are linked together,as in RNA, they are enhanced in concert. In fact, the relative intensities are very similar to a normal Raman spectrum of E.coli RNA, which shows the combined cytosine and uracil band at ca. twice the intensity of the adenine band, and ca. fourtimes the intensity of the guanine band. Unfortunately, this means that the independent enhancement factors for the NAbases can not be used to estimate relative concentrations. For example, the relative 791 and 734 cm-1 bands for B. subtiliswould indicate that the cytosine and/or uracil concentration was at least 20 times the adenine concentration, whereas each ofthe four RNA bases are known to contribute 15-35%. Nevertheless, it is worth noting that the three RNA samples yielddifferent relative band intensities that were reproduced in numerous measurements. Although the relative concentrations ofthe NA bases for these samples have not been determined, these differences can be quantified. If it is assumed that the 650cm-1 band represents 25% guanine, the 791 cm-1 band represents 25% adenine, and the 790 cm-1 50% cytosine plus uracil inthe E. coli RNA spectrum, then the relative concentrations can be estimated for the other RNA samples. To aid thiscalculation, the three spectra were normalized to the phosphate band at 1100 cm-1, which has been shown to correlate to thetotal phosphate concentration and can be used as an internal standard. In addition a simple baseline correction was applied(Figure 7). This yields 15% adenine, 30% guanine and 55% cytosine plus uracil for B. subtilis RNA and 18% adenine, 25%guanine and 57% cytosine plus uracil for S. aureus RNA. The average S/N of these measurements was 26 with an averageerror of 8% of the value (S±N).It is also worth noting that the three RNA spectra show a marked shift in a band near 825 cm-1. This band is assigned to thesymmetric stretch of the O-P-O ester linkage.9 The band appears at 815 cm-1 for S. aureus, shifting to 820 cm-1 for B. subtilis,and 830 cm-1 for E. coli. Others have used the normal Raman intensity of the band at 815 cm-1 as a direct indication of theamount of A-class helix present, while the intensity of the band at 830 cm-1 has been used as a direct indication of the amountof B-class helix present. However, the latter is more associated with DNA, than RNA.SPIE 2001-4575 68
  15. 15. A B OPO E.coli B. subtilis S. aureas G A C+U P Wavenumbers (∆cm-1) Wavenumbers (∆cm-1)Figure 7. SER spectra of RNA from A) B. subtilis with contributions indicated and B) E. coli, B. subtilis and S. aureuswith baseline correction and peak positions used to calculate % contributions indicated. G = guanine, A = adenine, C+U= cytosine plus uracil, P = phosphate (backbone), OPO = phosphate ester linkage (A- vs. B-class helix).SG-SER spectra of reasonable quality were also obtained for E. coli and B. subtilis, especially the latter (Figure 8). However,the spectra differ substantially from the E-SERS of the same samples. Both SG-SER spectra are dominated by adenine at735 cm-1 and a band at 1030 cm-1. Although unassigned, the latter does appear in the RNA E-SER spectra. Bands at 1105and 1565 cm-1 are likely due to phosphate, while bands at 1320 and 1470 cm-1 are less confidently assigned to phosphate.They are significantly less intense and somewhat shifted from their SG-SERS counterparts (1335 and 1455 cm-1). A band at670 cm-1 may be due to guanine, which was observed at 664 cm-1 for SG-SERS of the pure sample. However, the SG-SERspectrum of AMP also had an intense 667 cm-1 band. A number of other bands occur at 890, 1070, 1165, 1245, 1290, 1420,1505 cm-1 and remain unassigned. The SG-SER spectra are somewhat disappointing, in that only adenine and guaninecontributions can be positively identified. This limits the ability to determine relative NA base concentrations and distinguishbacterial RNA. However, several of the unassigned bands may be due to the bases (e.g. 1030 and 1420 cm-1 due to cytosine).Further experiments will be required to clarify this point. A B E-SERS E-SERS SG-SERS SG-SERS Wavenumbers (∆cm-1) Wavenumbers (∆cm-1)Figure 9. E-SER (-0.3VSCE) and SG-SER spectra of RNA from A) E. coli and B) B. subtilis. Sample conditions as inFigure 6. E-SER spectra at 750 mW, SG-SERS at 75 mW.SPIE 2001-4575 69
  16. 16. A final comparison was made between the two SER methods by measuring dipicolinic acid (DPA). This chemical may beinvaluable as a test for spore forming bacteria, specifically B. anthracis. 50 to 90% of B. anthracis sporilates. During sporeformation dipicolinic acid is synthesized, and once completed, 10-15% of the dry spore weight is composed of the Ca2+complex located in the spore core.42 Heating in water can be used to initiate germination, at which point the exosporiumbreaks and releases the Ca dipicolinate, which becomes dipicolinic acid in water. The structure of this chemical stronglysuggested that it would be SER active. However, the E- B (Ax20)SER spectrum was unstable and varied considerably as a A DPAfunction of potential. A consistent spectrum was obtainedat +0.6VSCE (Figure 10). This potential is notrecommended for measurement, because the surface isactively dissolving in solution. The SG-SER spectrum wasconsiderably more stable, of higher quality, and easilyreproduced. Bands at 660, 825, 1010, 1390, 1430, 1570, C1590, and 3075 cm-1 were observed. Enhancement factorswere determined for the two media using the symmetricring stretching mode at 995 and 1010 cm-1, for the normalRaman and SER spectra respectively. E-SERS yielded anEF of 5x103, while SG-SERS yielded an EF of 2x105 for DDPA. The S/N of the latter suggests a detection limit of2.0x10-10g (based on adenine coverage, 75 mW and 10-min). The differences in SER activity for these two mediamay be attributed to the combined electrolytic potential of Wavenumbers (∆cm-1)the solution, chemical and metal.15 Again the E-SERS Figure 10. A) Raman spectrum of solid dipicolinic acid,suggests that the SG-SERS is at a potential positive of the B) Ax20, C) electrolytic SERS of 6x10-3 M dipicolinicpzc. While the instability in the E-SERS may also be acid in 0.1 M KCl at a potential of +0.7VSCE and pH of 4,associated with surface interactions of two carboxylic acid and D) sol-gel SERS of 6x10-3 M dipicolinic acid.groups of dipicolinic acid during the ORCs. Conditions for A and C as in Figure 1, C) 100 mW of 1064 nm, 50 scans, 8 cm-1. 4. CONCLUSIONSTowards the goal of developing a method and technology to rapidly detect and identify bioagents, we have been investigatingsurface-enhanced Raman spectroscopy as a tool to measure relative concentrations of nucleic acid bases in RNA anddetermine bioagent taxonomy. Initially, we investigated E-SERS, since this method has been extensively researched, and theoptimum sample conditions are well developed. However, this method requires a three-electrode sample cell and electrolytesolution. Incorporation of an electrolytic cell into sample systems used to analyze for BWA as aerosols, in water or onsurfaces can be designed using flow injection analysis technologies, but cross-contamination and plugging of sample linesseems inevitable. For this reason, we also investigated metal-doped sol-gels as a SER-active medium. Previous studies haveshown this material to be active in all solvents, particularly water, capable of continuous measurements in flowing systems,and reproducible (quantitative) between coated sample vials. Here we compared SG-SER spectra to traditional E-SERspectra of the nucleic acid base pairs, adenosine monophosphate and RNA.High quality spectra of adenine, cytosine, guanine, thymine and uracil were obtained by both E-SERS and SG-SERS. Bothmethods yielded very similar spectra for the NA bases, including a pH dependent study of adenine. Enhancement factors anddetection limits for adenine were determined as 2x105 and 1.6x10-11g, and 1x105 and 1.2x10-10g for E-SERS and SG-SERS,respectively (normalized to 75 mW and 10-min acquisition time). Fifty percent of the silver particle surface area in the sol-gel matrix was assumed covered by adenine, which may have been overestimated yielding a lower EF and higher detectionlimit. It should also be realized that each E-SER spectrum required several attempts to optimize the measurement conditions(pH, electrode potential, etc.). While each SG-SER spectrum involved no sample preparation, and often represents the firstand only attempt to make the measurement.Quality spectra of RNA extracted from Escherichia coli, Bacillus subtilis and Staphylococcus aureus were obtained by E-SERS that were easily interpreted. Bands due to adenine, guanine, cytosine plus uracil, and phosphate were identified. TheSER band intensity of the NA bases in the RNA samples were of the same order of magnitude, suggesting that theirinteraction with the silver surface is concerted as is their Raman enhancement. Interestingly, the relative SER bandSPIE 2001-4575 70
  17. 17. intensities for RNA extracted from E. coli are very similar to those measured by normal Raman spectroscopy. Although therelative percent that each of the NA bases contributed to each RNA sample was not determined, reproducible band intensitiesallowed noting the following trends. The percent adenine decreases, while the combined percent cytosine and guanineincrease for both B. subtilis and S. aureus compared to E. coli. Quality spectra were also obtained for the RNA samples bySG-SERS, but only a few bands were readily identified. Calculations of NA base concentrations by SG-SERS will requirefurther research.In light of recent events, we summarize the capabilities of these SERS media in terms of rapid detection of B. anthracis anddipicolinic acid. However, these capabilities must be qualified. First and foremost, the level to which SERS can distinguishbacteria or viruses has not yet been determined. Development of a database of both DNA and RNA base concentrations forBWAs and common bacteria to establish the level of taxonomic identification is ongoing. Second, rapid collection ofaerosol, water, or surface samples is being addressed by others, who report trapping particles on filters from 100 liters of airper minute. Third, although not presented here, we have developed methods to extract RNA or DNA from cells and sporesfor SER analysis within 10 minutes. Finally, we assume a detection limit of 3600 spores per 100 liters of air is required,although a 50% lethal dosage of anthrax has not been established. With these qualifications, a mass detection limit for RNAusing SERS is estimated as follows. A single measurement is performed in ca. 20 minutes (140 liters collected in 1.4 min,RNA extracted in 8 min, spectral acquisition and analysis in 10 min). The average human breaths 7 liters per minute,therefore the analyzer must, at the very minimum, detect 5000 spores in 140 liters of air. One spore is approximately2x10-18m3 (1x1x2 µm), and if a density of 0.75 g/cm3 is assumed, this corresponds to a mass of 1.5x10-12g. Each sporecontains 4-12% RNA or 1.2x10-13g RNA for 8%. If we assume 2/3 of the RNA can be isolated for analysis during lysis, thenthe proposed instrument must be able to detect 4x10-10g RNA from 5000 spores per 70 liters of air within 10 minutes. Asnoted above, the mass detection limits for adenine were estimated at 1.6x10-11g, and 1.2x10-10g for E-SERS and SG-SERS,respectively. Although, these detection limits suggests that RNA from 5000 spores is detectable with the currentinstrumentation, it is highly likely that only a portion of an RNA segment (e.g. 120-nucleotide 5S rRNA) is in contact withthe metal surface and will contribute to the SER effect. The S/N for the RNA spectra were 1/10th of the average S/N for thefour individual RNA bases suggesting a 10% contribution. Furthermore, effective taxonomy will likely require knowing theNA base concentrations to 1% of the value (e.g. 25±0.25%). Again the average measurement error for the bases is 12%.These values suggest that the E-SERS is within a factor of 4 of the required detection limit, whereas the SG-SERS detectionlimit must be improved by 25 times.The same arguments can be applied to the detection of dipicolinic acid. If we assume a spore releases 10% by weight DPAduring germination, then the proposed instrument must be able to detect 7.5x10-10g DPA from 5000 spores per 70 liters of airwithin 10 minutes. The detection limit for SG-SERS was estimated at 2.0x10-10g and suggest that the vials are suitable toperform a rapid screen for anthrax. A series of concentration dependent measurements are currently being performed toverify this assertion.Finally, we note that the measurements performed here employed an FT-Raman spectrometer. This instrumentation waschosen over dispersive Raman instrumentation for the high wavelength accuracy afforded by the HeNe reference laser(Connes Advantage43), which would allow reliable spectral subtraction, matching of observed spectra to stored libraryspectra, and confident use of chemometric approaches. Such data analysis is likely to be required to enhance BWAidentification. However, this instrumentation sacrifices sensitivity due to a less efficient detector (InGaAs vs. Si), lessefficient Raman scattering, and less efficient generation of plasmon modes. Substantial improvements can be obtained usingvisible excitation and Si detection and these measurements are underway. 5. ACKNOWLEDGEMENTSThe authors are grateful to Drs. D. Cookmeyer and S. Tove of the U.S. Army Research Office (Contract Number DAAH04-96-C-0078) for their interest and support of this research. The authors would also like top acknowledge Dr. R. Yin and J.Jensen for supporting development of the metal-doped sol-gels (Contract Number DAAD13-01-C-0019). They also thankDr. Wilfred H. Nelson for assistance in spectral interpretations.SPIE 2001-4575 71
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  19. 19. Appendix C Chemical agent identification by surface-enhanced Raman spectroscopy Stuart Farquharson* and Paul Maksymiuk Real-Time Analyzers, 87 Church Street, East Hartford, CT 06108 Kate Ong and Steven D. Christesen U.S. Army, SBCCOM, Aberdeen Proving Ground, MD 21010 ABSTRACTThe recent distribution of anthrax through the U.S. postal system and the subsequent infection and death of several postal andnational media employees, amplifies the need for methods to rapidly detect, identify, and quantify this and other chemicaland biological warfare agents. The U.S. military has also identified water supplies as a likely method of warfare agentdeployment and is funding the development of a Joint Service Agent Water Monitor (JSAWM). In an effort to aid militarypersonnel and the public at large, we are developing a portable analyzer capable of identifying and quantifying chemicalagents rapidly, either "on-demand" or continuously. The approach is based on the ability of Raman spectroscopy to identifymolecular structure through the abundant vibration information provided in spectra and the ability of surface-enhancedRaman spectroscopy (SERS) to detect extremely low concentrations (e.g. part-per-billion) through the enhancement ofRaman scattering by six orders of magnitude or more. A key element to the analyzer design is a new SER active mediumthat is capable of quantitative, reversible measurements. The medium consists of silver or gold nanoparticles incorporatedinto a sol-gel matrix. The porous silica network offers a unique environment for stabilizing SER active metals and the highsurface area increases the interaction between the analyte and metal particles. Here we present the use of new sol-gels thatalso selectively enhance chemicals based on polarity and charge. Base-line measurements of chemical agents and theirhydrolysis products are presented and compared to the JSAWM goal of 3.0 micrograms per liter detection.Keywords: Chemical warfare agent, hydrolysis product, SERS, Raman spectroscopy, sol-gel, nanoparticle 1. INTRODUCTIONSince September 11, 2001, the threat of terrorist attacks and biological warfare within U.S. borders has become a soberingreality. The simplicity in manufacturing, ease of deployment, and the relatively low cost of chemical warfare agents (CWAs)raises public concern that they may also be used by terrorists. Indeed, terrorists released sarin (GB) in the Tokyo subway in1995.1 Countering terrorism and terrorist attacks requires recognizing likely deployment scenarios and having the requiredtechnology to rapidly detect the deployment event. One method of deployment has been long identified by the U.S. military:distribution through water supplies. To counter this threat, the Department of Defense is funding or monitoring thecapabilities of 200 technologies with the goal of developing a Joint Service Agent Water Monitor (JSAWM) that is fieldportable.2 The JSAWM must be capable of identifying the chemical agents with no false-positives and quantifying the agentsat microgram per liter concentrations within 10 minutes (Table 1).3 This includes the analysis of drinking water supplies,distribution and storage systems, as well as potable water supplies.The earliest technologies developed for CWA detection were based on electrochemical, ionization, or colorimetric analysis(e.g. phosgene tape). Although these analyzers were easy to use, they were not generally agent specific and suffered fromfalse-positives.4 More traditional laboratory methods have also been investigated, and in particular, combined gaschromatography and mass spectrometry (GC/MS) has been very successful at eliminating false-positives.5,6 However,GC/MS requires extraction, repeated calibration, and long analysis times (typically 20 to 60 minutes),6 making it laborintensive and less than desirable for field use. More rapid analysis of agents in the solid, liquid and gas phase has been* To whom correspondence should be addressed, email:farqu@real-time-analyzers.com Vibrational Spectroscopy-based Sensor Systems, Steven D. Christesen, Arthur J. Sedlacek III, Editors,166 Proceedings of SPIE Vol. 4557 (2002) © 2002 SPIE ·0277-786X/02/$15.00
  20. 20. Table 1. Chemical Agent Structures, Hydrolysis Half-lives, and JSAWM Thresholds. Agent Short-Hand Chemical Structure Hydrolysis JSAWM Half-Life* Thresholds Sarin (GB) F-[O=P-CH3]-O-CH(CH3)2 21.3 hours 3.2 µg/L Soman (GD) F-[O=P-CH3]-O-CH(CH3)-(C-(CH3)3) 2.3 hours 3.2 µg/L Tabun (GA) (CH3)2-N-[O=P-CN]-O-C2H5 4.1 hours 3.2 µg/L VX C2H5O-[O=P-CH3]-S-(CH2)2-N-(CH(CH2)2)2 82.1 hours 3.2 µg/L EA2192 HO-[O=P-CH3]-S-(CH2)2-N-(CH(CH2)2)2 >9 years 3.2 µg/L Mustard (H) ClCH2CH2-S-CH2CH2Cl encapsulates 47 µg/L Lewisite (L) ClCH=CH-As-Cl2 rapid 27 µg/L HCN HCN rapid 2.0 mg/L BZ** C7NH12-O-[C=O]-COH(C6H5)2 2.3 µg/L T-2 Toxin 8.7 µg/L * at pH 7 to 7.5 and 20 to 25 oC.demonstrated by vibrational spectroscopy.7-10 Hoffland et al.7 reported infrared absorbance spectra and absolute Raman crosssections for several chemical agents, while Christesen measured Raman cross sections for sarin, tabun, mustard gas, and VX(ethyl S-2-diisopropylamino ethyl methylphosphonothioate).11 Again, however these techniques also have limitations.Raman spectroscopy is simply not a very sensitive technique, and detection limits are typically 0.1% (1000 ppm). Whileinfrared spectroscopy would have limited value in analyzing poisoned water, since the very strong infrared absorption ofwater would obscure most other chemicals present. Nevertheless, efforts to overcome these limitations have beendemonstrated. Braue and Pannella8 quantified the G-series nerve agents (tabun, sarin, and soman) in terms of infraredattenuated total reflectance using a circle-cell. And Alak and Vo-Dinh demonstrated the possibility of surface-enhancedRaman spectroscopy (SERS) to identify CWAs by measuring several organophosphonates that simulate the nerve agents.12However, quantitative measurements have not been demonstrated for the SER-active material used (silver coated on aluminaparticles) or other SER-active media.13Recently, we developed silver-doped sol-gels to promote the SER effect.14-17 The porous silica network of the sol-gel matrixoffers a unique environment for stabilizing SER-active metal particles, and the sol-gel provides a high surface area thateffectively increases the number of molecules observed within the Raman scattering volume. The silver-doped sol-gels havebeen coated on the internal walls of standard glass vials to allow rapid SER analysis of small sample quantities (< 0.1 mL)without preparation. We have used p-aminobenzoic acid (PABA) as a test chemical to demonstrate surface enhancementsgreater than 106, reversible measurements in a flowing system, reproducible measurements from vial-to-vial and batch-to-batch, and measurements in multiple solvents, including water.14-17 Recently, we used these vials to measure Tabun (GB) andSarin, and several hydrolysis products, pinacolyl methyl phosphonate (PMP from Soman), and methyl phosphonic acid(MPA from all G-agents, Figure 1). Although a number of unique vibrational bands are observed (e.g. C-N stretch doubletand P-C stretch), the G-agents were only observed for 5% concentrations, and all spectra required baseline corrections. A C 790 C-N P-C 2135, 2190 770 545 1290 B D 760 Wavenumber (∆cm-1) Wavenumber (∆cm-1)Figure 1. Surface enhanced Raman spectra of ~5% v/v A) Tabun and B) Sarin, C) 1% v/v PMP and D) 10 ppm MPAusing sol-gel sample vials, 785 nm excitation, 1-min scan, and CCD detection. Performed at Aberdeen Proving Ground. Proc. SPIE Vol. 4577 167
  21. 21. Nevertheless, MPA was readily observed for a 10 ppm sample, with an estimated detection limit of 0.4 ppm (based on asignal-to-noise ratio of 3 for the 760 cm-1 band intensity). This measurement provides encouragement in that SERS maysatisfy the needs of a JSAWM. Furthermore, MPA is also a hydrolysis product of VX and V-gas, and EA2192 (Figure 2),and may prove a valuable indicator of agent usage. O CH3 O CH3 O CH3 P C + H 2O HF + P C P + C H3C O CH3 H3C O CH3 H3C OH HO CH3 F OH OH Sarin MPAMME MPA 2-propanolFigure 2. Hydrolysis of Sarin to form hydrofluoric acid (HF), methylphosphonic acid, 1-methylethyl ester (MPAMME),methyl phosphonic acid (MPA) and 2-propanol.With this initial, albeit modest, success, we began analyzing chemicals with various sol-gel compositions that we have beendeveloping. Here we describe four sol-gel compositions that select for 1) polar-positive, 2) polar-negative, 3) weakly polar-positive and 4) weakly polar-negative chemical species. The ability of these sol-gels to select and enhance Raman scatteringis described for several test chemicals and MPA. 2. EXPERIMENTALThe chemicals analyzed, as well as all chemicals used to prepare the metal-doped sol-gels were obtained at their purestcommercially available grade from Aldrich (Milwaukee, WI). The sol-gel designed to select for polar-negative species wasprepared from a silver amine complex, tetramethyl orthosilicate (TMOS) and methanol. After mixing, 0.2 mL of the sol-gelsolution was transferred into a glass vial (2 mL), dried and heated. The incorporated silver ions were then reduced usingdilute sodium borohydride. The vials were washed and dried prior to the addition of a sample solution. In a similar manner,the sol-gel designed to select for polar-positive species was prepared from a gold salt, TMOS and methanol. The sol-geldesigned to select for weakly polar-negative species was prepared from a silver amine complex, tetraethyl orthosilicate(TEOS) and methanol. And the last sol-gel designed to select for weakly polar-positive species was prepared from a goldsalt, TEOS and methanol.All samples were prepared in a chemical hood and transferred into plain or SER-active vials for analysis. Normal Ramanspectral measurements employed 1-mL pure samples that were placed in a 1-cm3 cuvette and weighed. This yielded apowder density that allowed accurate calculation of molecules in the optical collection field. SERS measurements employed1-mg sample per mL water concentrations, unless otherwise stated. Once prepared, a 0.1 mL sample was placed into one ofthe four selective sample vials, which in turn was placed into the sample compartment of a Raman spectrometer for analysis.A prototype Fourier transform Raman spectrometer (RTA) was used for these measurements.18 The system consisted of aNd:YAG laser (Brimrose) for excitation at 1064 nm, an interferometer built by On-Line Technologies (OLT, East Hartford,CT) for frequency separation, an uncooled InGaAs detector for signal detection (RTA), and an Intel 400 MHz Pentium IIbased laptop computer (Dell, Round Rock, TX) for interferometric control, data acquisition (OLT), and analysis (LabVIEWby National Instruments, Austin, TX). Additional components included a Notch filter (Kaiser, Ann Arbor, MI) andinterferometer entrance and exit optics (Edmund Scientific, Barrington, NJ). Fiber optics were used to deliver the excitationbeam to the sample and the scattered radiation to the interferometer (1 meter lengths of 200 and 365 micron core diameter,respectively, Spectran, Avon, CT). A second Notch filter (Kaiser) was used as a beam splitter to direct the excitation beamalong the same axis as the collected radiation. A microscope object (20x0.4, Newport, Irvine, CA) was used to focus thebeam into the sample and to collect the scattered radiation back along the same axis. In this co-axial backscatteringarrangement, the excitation beam was passed through the outside of a glass vial and focused onto the silver-doped sol-gelfilm (0.1-0.3 mm thickness) containing the sample. 3. RESULTS AND DISCUSSIONp-aminobenzoic acid (PABA) and phenyl acetylene (PA) and were used to refine the selectivity and SER-activity of the fourdifferent metal-doped sol-gels. PABA is a popular chemical used to evaluate the performance of SER-active media. Herethe polar end groups can be used to test selectivity of the polar-negative and polar-positive sol-gels. PA is essentially non-168 Proc. SPIE Vol. 4577

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