Monitoring DPA Release from a Single Germinating Bacillus subtilis Endospore via Surface-Enhanced Raman Scattering Microscopy David D. Evanoff, Jr., John Heckel, Thomas P. Caldwell, Kenneth A. Christensen, and George Chumanov* Department of Chemistry, Clemson UniVersity, Clemson, South Carolina 29634 Received June 16, 2006; E-mail: gchumak@clemson.edu During the past decade there has been a great deal of emphasis placed on developing reliable and rapid methods for the detection of Bacillus anthracis, the causative agent of anthrax. The Center for Disease Control estimates the inhalation LD 50 of B. anthracis to be on the order of 100 ng, or about 10 4 spores. 1 Methods such as staining, immunoassays, polymerase chain reaction (PCR), 2 electrophoretic, and chromatographic techniques 3 are all quite capable of detecting B. anthracis, and in some cases differentiating Bacillus species, well below the human LD 50 . However, since these methods generally take on the order of hours to complete, appropriate interventions are delayed. Spectroscopic techniques such as photoluminescence, FT-IR, Raman, and CARS 4 have also successfully been used to identify Bacillus endospores through the detection of the acidic and/or calcium-chelated dipicolinate ion (DPA). Advantageously, DPA represents ca. 10% of the total spore weight and is not found in other common spores such as pollen or mold. Extraction of DPA using dodecylamine 5 or nitric acid 6 can be followed by direct measurement using vibrational spectroscopy or photoluminescence of terbium after complexation with DPA. Raman spectroscopy can be used for the direct detection of DPA as it exists inside the spore. These measurements were first reported in 1974 for B. megaterium 4c and were recently used to detect relatively small amounts of B. cerus on a mail sorting system. 1 Raman microspectroscopy has been used to detect DPA contained in a single Bacillus endospore. 7 Several orders of magnitude of signal enhancement as well as reduction of the excitation power and spectral collection time can be obtained with surface enhanced Raman scattering (SERS) spectroscopy. 5,6,8 A recent report from this laboratory detailed the first study of the DPA release kinetics of B. subtilis (an anthrax stimulant) using SERS spectroscopy. 9 Germination was initiated by the natural germinant L-alanine and was monitored by the increase in DPA signal over time. The DPA release kinetics were measured as a function of L-alanine concentration and temperature and averaged over several hundred spores. Understanding the kinetics of DPA release can assist in determining a mechanistic picture of how L-alanine and other germinants initiate the transformation of endospores to vegetative cells. This level of biochemical under- standing is an important step for developing new B. anthracis therapeutics. In this communication, we extend this method for monitoring the DPA release from a single germinating B. subtilis endospore. High S/N ratio SERS spectra were obtained with excitation power as low as 3 mW and 1 min spectral collection times. Previous reports measured DPA localized within a single spore. 10 The current method detects only the signal of DPA that is released during the beginning of the germination process owing to the extreme surface localization of the SERS phenomenon. Hence, this method is selective for actively germinating spores. B. subtilis endospores and SERS-active substrates were prepared as previously reported. 8a,9 The sandwich-type SERS substrates were fabricated by assembling 100 nm Ag particles on poly(diallyldi- methylammonium chloride) (PDDA) modified silver mirror film (Figure 1A). A typical surface density of the particles was 12-15 μm -2 . The assembled particles were further modified with PDDA rendering a positive surface charge and providing adsorption of the negatively charged spores to the substrate. The substrates were exposed overnight to a dilute spore suspension to obtain low surface coverage of the spores. The average spacing between spores from which the SERS was measured was 30 μm ensuring that the collected spectra originated from a single endospore without the interference from DPA released by neighboring spores. Also, no DPA signal was detected from the substrate alone ca. 10 μm away from a germinating spore further indicating that the SERS signal was localized to the proximity of the spore. This localization resulted from trapping negatively charged DPA molecules by the layers of the positively charged PDDA polymer. The spores were imaged in reflection geometry because of the opaque nature of the SERS substrate (Figure 1B). Before the SERS measurement, 10 μL of a 150 mM L-alanine solution was dropped onto the substrate with adsorbed spores and sealed using a coverslip and vacuum grease. Individual spores were located, and spectral collection was initiated within 1-2 min after the L-alanine addition. Imaging and spectral acquisition was done using a 100×/1.3 N.A. oil immersion objective on an Olympus inverted microscope equipped with a Raman spectrograph and liquid nitrogen cooled CCD camera. The 647.1 nm line from a Kr + laser was used for excitation. To characterize the germination kinetics, a series of SERS spectra were collected at 1.5 min intervals over 30 min time period. Each spectrum was accumulated during a 60 s interval followed by a 30 s waiting period between the scans. All spectra measured from the same spore were normalized to a broad band of PDDA 9 at 794 cm -1 . This band served as an internal standard for the quantitative comparison of SERS spectra acquired at Figure 1. (A) Schematic of the SERS substrates with an adsorbed spore and (B) a brightfield image of a single B. subtilis endospore on a SERS- active substrate at 100× magnification taken in reflection mode. Published on Web 09/09/2006 12618 9 J. AM. CHEM. SOC. 2006, 128, 12618-12619 10.1021/ja0642717 CCC: $33.50 © 2006 American Chemical Society