Surface-enhanced Raman scattering (SERS) detection for chemical and biological agents

Fei Yan, David L. Stokes, Musundi B. Wabuyele, Guy D. Griffin, Arpad A. Vass, Tuan Vo-Dinh* Advanced Biomedical Science and Technology Group Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831-6101

ABSTRACT

Surface-enhanced Raman scattering (SERS) spectra of chemical and biological agent simulants such as dimethyl methylphonate (DMMP), pinacolyl methylphosphonate (PMP), diethyl phosphoramidate (DEPA) and 2-chloroethyl ethylsulfide (CEES), bacillus globigii (BG), erwinia herbicola (EH), and bacillus thuringiensis (BT) were obtained from silver oxide film-deposited substrates. Thin AgO films ranging in thickness from 50 nm to 250 nm were produced by chemical bath deposition onto glass slides. Further Raman intensity enhancements were noticed in UV irradiated surfaces due to photo-induced Ag cluster formation, which may provide a possible route to producing highly useful probes for the detection of chemical and biological agents upon visible illumination.

Keywords: Metallic nanostructures, surface-enhanced Raman scattering, chemical simulants, biological simulates, CWA, silver nanoclusters, bioanalysis

1. INTRODUCTION

Concepts based on Raman scattering have frequently been proposed for remote or stand-off detection of chemical and biological agents. Raman spectroscopy is based on vibrational transitions that yield very narrow spectral features which are characteristic of the investigated sample. Thus, it has long been regarded as a valuable tool for the identification of chemical and biological samples as well as the elucidation of molecular structure, surface processes, and interface reactions. Despite such advantages, normal Raman scattering suffers the disadvantage of extremely poor efficiency. Compared to luminescence-based processes, Raman spectroscopy has an inherently small cross-section (e.g. 10-30 cm2 per molecule), thus precluding the possibility of analyte detection at low concentration levels without special enhancement processes. Some modes of signal enhancement have included resonance Raman scattering and nonlinear processes such as coherent anti-Stokes Raman scattering. However, the need for high-power, multiple- wavelength excitation sources has limited the widespread use of these techniques.1 Hence, remote normal Raman detection of chemical agents was considered to be unreliable.2 Because of the aggressive development of SERS substrates and application to a wide range of chemicals, the potential of SERS as a routine analytical technique was recognized by the mid-1980s. More recent reports have cited SERS enhancements from 1013 to 1015, thus demonstrating the potential for single-molecule detection with SERS. 3-9

In this paper, we report the detection of chemical and biological agent simulants using a type of SERS- active substrate based on chemically deposited silver oxide films, and demonstrate the Raman signal enhancement due to the silver cluster formation upon visible illumination.

2. FORMATION OF SILVER OXIDE THIN FILMS AND NANOCLUSTERS

Chemical bath deposition (sometimes called solution growth or electroless deposition) is the simplest way to deposit thin films of some metal oxides, which are known to have spectrally selective characteristics suitable for photothermal and material applications.10 Uniform and specularly reflective silver oxide films

*To whom correspondence should be addressed. E-mail: [email protected]. with different thickness were produced on glass substrates using methods reported in a prior study.11 Briefly, 1 g of solid AgNO3 was dissolved in 5 mL of deionized water in a 100-mL beaker. Then, triethanolamine solution was added gradually, with constant stirring, until the initially formed precipitate was dissolved. More deionized water was added to make a total volume of 80 mL. Pre-cleaned glass slides were vertically placed into the beaker and the bath was brought to and kept at 45-50 0C on a hot plate. After various periods of time (i.e., 5, 10, 15, 30 minutes), the coated slides were removed from the bath, thoroughly rinsed with deionized water and air-dried.

Silver oxide has a band gap in the visible region (2.25 eV or ~ 550 nm) 10 and is readily photoreduced to yield metallic Ag clusters. 12 It was further demonstrated that thin AgO films were readily photoactivated at 13,14 room temperature to produce dynamic, multicolored fluorescent Agn nanoclusters. Illumination of silver oxide samples with blue and UV mercury lamp excitation produces Agn nanoclusters that range in size from n = 2 to 8 atoms with strong size- and geometry- dependent fluorescence. 13 In this study, we photo-treated the silver oxide films chemically deposited on glass slides using a UV mercury lamp for different time periods, and examined their surface features by AFM before using them for SERS measurements.

3. CHARACTERIZATION OF SILVER OXIDE THIN FILMS AND SILVER NANOCLUSTERS

An explorer SPM model AFM (ThermoMicroscopes, Sunnyvale, CA) was used for imaging of the surfaces. The AFM was operated in the non-contact mode, and measurements were performed under ambient conditions. Silicon tips with a resonance frequency of 240-420 kHz and a force constant of 40 N/m were used.

A B

C D

Figure 1. AFM images of silver oxide films grown on glass slides by a chemical-bath-deposition technique. A) 5 min, B) 10 min, C) 15 min, and D) 20 min.

Fig. 1 demonstrates the gradual increase of silver oxide film thickness with increasing deposition time. An average surface height of 49.61 nm, 118.08 nm, 134.48 nm, 234.52 nm was measured for a deposition time of 5, 10, 15, 20 min, respectively. These results agree very well with the thickness estimated gravimetrically, assuming the same density as that of a bulk AgO or Ag (7.44 g/cm3 for AgO and 10.5 g/cm3 for Ag). 11

A B

C D

Figure 2. AFM images of silver oxide films grown on glass slides by a chemical-bath-deposition technique. A) before UV irradiation, after UV irradiation for B) 5 min, C) 15 min, and D) 30 min.

As shown in Fig. 2, the surface features of silver oxide film changes slightly upon UV illumination for 5 and 15 min, and more pronounced changes were observed after 30 min irradiation. The formation of Ag nanoclusters was more noticeable as demonstrated in Fig. 3.

Figure 3. Scanning electron micrograph of silver nanoclusters formed upon UV illumination, from a silver oxide film grown on a glass slide by a chemical-bath-deposition technique.

4. SURFACE-ENHANCED RAMAN SCATTERING STUDIES

4.1. SERS Detection of Chemical Agent Simulants

The SERS spectra were obtained on a conventional Raman spectrometer (Fig. 4). In this system, 632.8 nm radiation from a Helium–Neon laser is used with an excitation power of ~5 mW. Signal collection is performed at 0° with respect to the incident laser beam. This coaxial excitation/collection geometry was achieved with a small prism, which was used to direct the excitation beam to the sample while allowing most of the collected signal to pass on to the collection fiber. A Raman holographic filter is used to reject the Rayleigh scattered radiation from the collected Raman signal. The Raman signal is focused onto the entrance slit of a spectrograph (ISA, HR-320), which is equipped with a red-enhanced intensified charge-coupled device (CCD) detector (Princeton Instruments, RE-ICCD-576S), with a total accumulation time of 10 s per spectrum.

Coupling Optics Mirror

RE-ICCD Spectrograph Holographic Filter

Laser Line Filter Dichroic Mirror 632.8- nm He-Ne Laser

Objective Lens 60x PC XYZ Translation Stage Metallic Nanostructured Substrate

Figure 4. Schematic of the instrument setup used for SERS detection of chemical and biological agent simulants.

50000 DMMP 40000 30000

20000 SERS Intensity Intensity SERS (Arbitrary Units) (Arbitrary 10000

0 200 400 600 800 1000 1200 1400 A Raman Shift (cm-1)

300000 CEES 240000

180000

120000 SERS Intensity SERS (Arbitrary Units) (Arbitrary 60000

0 200 400 600 800 1000 1200 1400 Raman Shift (cm-1) B Figure 5. SERS spectra of chemical agent simulants on silver oxide film grown on a glass slide for 10 min: A) DMMP (1 ppm in water), and B) CEES (1 ppm in ethanol).

SERS spectra of DMMP and CEES are shown in Fig.5. Two main spectral features are clearly seen in the SERS spectra of all simulants under study. Despite the structural similarity among DMMP, PMP and DEPA, minor differences could still be resolved in the spectral region of our current Raman setup, especially for DEPA and CEES. The peaks at around 720 cm-1 for DMMP, PMP and DEPA correspond to a stretching mode involving the phosphorus atom, and a much stronger line at around 700 correspond to C- Cl and C-S stretches in SERS spectrum of CEES. Given the wavelength dependence of the Raman scattering cross section of all the chemical simulants, 12 it may be feasible to differentiate each of them if lasers of different wavelength were to be utilized for excitation. Fig. 6 illustrated an additional SERS signal enhancement due to the formation of Ag nanoclusters photo-produced from a silver-oxide thin film upon UV illumination. In this case, 15 minutes UV illumination resulted in ~50% Raman signal increase.

80000 CEES 70000

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30000 400 600 800 1000 1200 1400 1600 -1 Raman Shift (cm )

Figure 6. SERS spectra of CEES (1 ppm in ethanol) on Ag silver oxide film grown on a glass slide before (bottom) and after (top) UV illumination for 15 min.

4.2. SERS Detection of Biological Agent Simulants

Routine identification of pathogenic microorganisms is largely based on nutritional and biochemical tests. Although these techniques can sometimes provide results rapidly, they are relatively expensive and require highly skilled personnel.15 Recently, SERS has been shown to have great potential as a complementary whole-organism fingerprinting approach for the rapid characterization of bacteria.16 Initial SERS measurements of biological agent simulants on silver oxide thin films were not successful (data not shown). However, a SERS signal (Fig. 7) was obtained after the substrates were illuminated by an UV lamp for only 15 min. While there is the possibility of gaining structural information from these SERS spectra, they are difficult to interpret. There could be any number of SERS-active vibrational modes present in biochemically complex samples such as bacteria. In this case, with a priori information on the biochemistry of the organisms studied, it is possible to target investigations into certain peaks; however, at best any peak assignments can only be tentative. While many researchers have explored SERS for the characterization of bacteria, few of these studies has shown that bacteria can be discriminated by their SERS spectra. However, one recent study demonstrated the potential for SERS to produce spectra that could be used for rapid whole-organism fingerprinting, and in conjunction with simple cluster analyses, such hyperspectral data could be used to discriminate between microorganisms to the strain level.16 Similar research efforts are currently being undertaken in our group, and the findings will be published elsewhere.

40000 EH

35000

) 30000

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10000 600 700 800 900 1000 1100 1200

Raman Shift (cm-1)

Figure 7. SERS spectrum of erwinia herbicola on Ag silver oxide film grown on a glass slide after UV illumination for 15 min.

5. CONCLUSIONS

In summary, we demonstrated an alternative approach of preparing SERS-active substrates based on the formation of silver oxide films and /or Ag nanoclusters, and were able to obtain promising results from the detection of chemical and biological agent simulants. Knowledge obtained from these and future studies will definitely be very useful for the detection of real chemical and biological agents.

ACKNOWLEDGMENTS

This research was sponsored by the U.S Department of Energy (DOE) Office of Chemical and Biological National Security and the DOE Office of Biological and Environmental Research, under contract DEAC05- 000OR22725 with UT-Battelle. Fei Yan, David L. Stokes and Musundi B. Wabuyele are also supported by an appointment to the Oak Ridge National Laboratory Postdoctoral Research Associates Program, administered jointly by the Oak Ridge National Laboratory and Oak Ridge Institute for Science and Education.

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