Discovery of a Significant Optical Chromatographic Difference

Anal. Chem. 2006, 78, 3221-3225

Discovery of a Significant Optical Chromatographic Difference between Spores of Bacillus anthracis and Its Close Relative, Bacillus thuringiensis Sean J. Hart,* Alex Terray,† Tomasz A. Leski,‡ Jonathan Arnold, and Rhonda Stroud§

Chemistry Division, Bio/Analytical Chemistry, Naval Research Laboratory, Code 6112, 4555 Overlook Avenue, S.W., Washington, D.C. 20375

A significant difference between two closely related Bacillus spores has been discovered using optical chromatography. This difference can be harnessed for the separation of microscopic particles using opposing laser and fluid flow forces. Particles of different size, composition, and shape experience different optical and fluid forces and come to rest at unique equilibrium positions where the two forces balance. Separations in excess of 600 µm have been observed between Bacillus anthracis Sterne strain and its genetic relative, Bacillus thuringiensis. These findings open new possibilities for detection and characterization of the biological warfare agent, B. anthracis, the causative agent of anthrax, the deadly mammalian disease. The large optical separation between these species is surprising given their close genetic relationship but may be explained by differences in their shape and exosporium morphology, which may result in differences in fluid drag force. The observation of large differences due to less common variables indicates the complex nature of the force balance in optical chromatography, which may in the future be used to separate and characterize microbiological samples. In general, the discovery of such large differences between such closely related biological species suggests new possibilities for the separation and characterization of microorganisms using the full range of emerging techniques that employ radiation pressure (optical filtering, laser tweezers, optical chromatography, etc.). Light from a laser can be used to exert significant forces on microscopic particles resulting from the transfer of photon momentum to matter.1 Laser radiation pressure has been used to trap and propel particles in a liquid, micromanipulate, form patterns, and study colloidal particles in numerous applications.2-8 In addition, there has been much interest in using optical forces * Corresponding author. E-mail: [email protected]. Tel: (202) 404-3361. Fax: (202) 404-8119. † Current address: SAIC, 1220 12th St., SE Suite 140, Washington, DC 20003. ‡ National Research Council (NRC) postdoctoral fellow. § Electronic and Optical Materials and Devices, Naval Research Laboratory, Code 6364, 4555 Overlook Ave., S. W., Washington, DC 20375. (1) Ashkin, A. Phys. Rev. Lett. 1970, 24, 156-159. (2) Ashkin, A.; Dziedzic, J. M.; Yamane, T. Nature 1987, 330, 769-771. 10.1021/ac052221z CCC: $33.50 Published on Web 03/28/2006

© 2006 American Chemical Society

to separate inorganic, polymeric, and biological colloidal samples.9-11 Optical trapping has been used for the manual sorting of particles based on appearance. Recently, more sophisticated and automated optical techniques have been developed to perform decision-based separations of microscopic particles based upon discernible features (size, shape, color, or fluorescence).12 Other methods have relied on intrinsic properties to effect colloidal separations. One such technique employed a mildly focused laser beam perpendicular to a fluid flow to selectively remove particles if the optical force were sufficient to propel them out of the flow profile.11 More recent efforts involve arrays of optical traps in a fluid flow to preferentially transport microscopic objects that experience a greater optical force away from those that experience a lesser force.9,10 Another innovative approach has been to use the optical features of a Bessel beam to sort particles in the absence of external fluid flow.13 Optical chromatography is a technique based upon intrinsic colloidal properties that has been used for the separation and analysis of microscopic particles based upon their physical and chemical characteristics.14-16 In this technique, radiation pressure resulting from a mildly focused laser beam accelerates particles against a fluid flow traveling in the opposite direction. When the (3) Bronkhorst, P. J. H.; Grimbergen, J.; Sixma, J. J.; Heethaar, R. M.; Brakenhoff, G. J. Prog. Biophys. Mol. Biol. 1996, 65, PH513-PH513. (4) Grier, D. G. Nature 2003, 424, 810-816. (5) Bustamante, C. Biophys. J. 2001, 80, 20A-21A. (6) Terray, A.; Oakey, J.; Marr, D. W. M. Science 2002, 296, 1841-1844. (7) Ashkin, A. IEEE J. Sel. Top. Quantum Electron. 2000, 6, 841-856. (8) Mio, C.; Gong, T.; Terray, A.; Marr, D. W. M. Rev. Sci. Instrum. 2000, 71, 2196-2200. (9) MacDonald, M. P.; Spalding, G. C.; Dholakia, K. Nature 2003, 426, 421424. (10) Korda, P. T.; Taylor, M. B.; Grier, D. G. Phys. Rev. Lett. 2002, 89. (11) Buican, T. N., Smyth, M. J., Crissman, H. A., Salzman, G. C., Stewart, C. C., Martin, J. C. Appl. Opt. 1987, 26, 5311-5316. (12) Oakey, J.; Allely, J.; Marr, D. W. M. Biotechnol. Prog. 2002, 18, 14391442. (13) Paterson, L.; Papagiakoumou, E.; Milne, G.; Garces-Chavez, V.; Tatarkova, S. A.; Sibbett, W.; Gunn-Moore, F. J.; Bryant, P. E.; Riches, A. C.; Dholakia, K. Appl. Phys. Lett. 2005, 87. (14) Hart, S. J.; Terray, A. V. Appl. Phys. Lett. 2003, 83, 5316-5318. (15) Kaneta, T.; Ishidzu, Y.; Mishima, N.; Imasaka, T. Anal. Chem. 1997, 69, 2701-2710. (16) Hart, S. J.; Terray, A.; Kuhn, K. A.; Arnold, J.; Leski, T. A. Am. Lab. 2004, 36, 13-17.

Analytical Chemistry, Vol. 78, No. 9, May 1, 2006 3221

optic and fluid forces are equal the particles remain stationary at their equilibrium position. The distance traveled from the focal point, the optical retention distance (Z), is characteristic of the particle’s size, shape, and refractive index, n (a measure of its chemical composition).14,15 Our initial goals are to find and understand the nature of the optical chromatographic separations between individual particle types, so that we may exploit these effects using a future bulk separation device currently under development. In this correspondence, we report the discovery of a significant optical chromatographic separation between spores of Bacillus anthracis (B.a.) and Bacillus thuringiensis (B.t.) (considered to be strains of the same species based on genetic evidence).17 B. anthracis, B. thuringiensis, and Bacillus cereus belong to a cluster of closely related species called the B. cereus group.18 Biochemical and genetic evidence obtained by multilocus enzyme electrophoresis and multilocus sequence typing suggests that these three species should be considered a single species.17 B.t. is a common spore that may interfere with the detection of B.a. since it can be found in a variety of environments: strains of B.t. have been found in such habitats as soil, insects, stored-product dust, and leaves. 19,20 Due to production of insecticidal proteins, numerous strains of B.t. are used as a pesticide and B.t. sprays currently comprise 2% of the global insecticide spray market,21 which strongly contributes to their widespread presence. The ability to distinguish B.a. from B.t. represents an important step toward developing optical methods for separating and detecting weaponized B.a. from complex environmental mixtures. EXPERIMENTAL SECTION Optics. In optical chromatography, a laser is lightly focused by a long focal length lens (50-100 mm) rather than a high numerical aperture objective (NA > 1.3), as used in optical trapping. The light is directed into a channel containing the microscopic particles suspended in a fluid traveling in the opposing direction. The system consisted of a Ti:sapphire laser tuned to 850 nm that was pumped by a CW 532-nm solid-state Nd:YVO4 laser (3900S and Millennia Xs, Newport-Spectra-Physics, Irvine, CA). The 850-nm laser beam was directed by mirrors to a 12.7mm-diameter, 25-mm-focal length aspheric lens (AC127-025-B, Thorlabs, Newton, NJ) mounted on an x-y-z translation platform (460XYZ, Newport Corp., Irvine, CA). The laser was aligned with the microfluidic network using a five-axis positioner (model 9081, New Focus, San Jose, CA) attached to an x-y-z translation platform (460XYZ, Newport Corp.). The fluidic network was coupled to the optomechanical components via a polycarbonate base fitted with fluidic connectors (Nanoport N-121S, Upchurch Scientific, Inc., Oak Harbor, WA) to couple both fluid and sample introduction tubing. (17) Helgason, E. Ø., O. A.; Caugant, D. A.; Johansen, H. A.; Fouet, A.; Mock, M.; Hegna, I.; Kolstø, A. B. Appl. Environ. Microbiol. 2000, 66, 26272630. (18) Sneath, P. H. A. Bergey’s Manual of Systematic Bacteriology; The Williams & Wilkins Co.L Baltimore, MD, 1986; Vol. 2, pp 1105-1139. (19) Martin, P. A. W.; Travers, R. S. Appl. Environ. Microbiol. 1989, 55, 24372442. (20) Schnepf, E. C. N.; Van Rie, J.; Lereclus, D.; Baum, J.; Feitelson, J.; Zeigler, D. R.; Dean, D. H. Microbiol. Mol. Biol. Rev. 1998, 62, 775-806. (21) Nester, E. W.; Metz, M.; Gordon, M. Am. Acad. Microbiol., Washington, DC 2002.

3222 Analytical Chemistry, Vol. 78, No. 9, May 1, 2006

Figure 1. Fluidic network showing the laser counter propagating against the liquid flow. Sample injection ports are located just downstream of the focal point.

The aligned optic and fluidic system was placed underneath a microscope (Multiscope, LOMO America, Inc., Prospect Heights, IL) that was mounted on an x-y-z translation platform (460XYZ, Newport Corp.). This allowed imaging of the entire flow cell under the microscope by translating the microscope, while not disturbing the optic and fluidic alignment. Images and video were obtained using image capture and analysis software (Simple PCI, Compix Inc., Cranberry Twp, PA) and a CCD camera (Retiga 1300C, Q-Imaging, BC Canada) coupled to the microscope. Microfluidics. Fluid flow was achieved using gravity feed and a syringe pump. The syringe pump was used for the occasional increase in flow required to rinse the flow cell while the gravity feed system was used to obtain extremely stable, pulseless flow. The flow rate from gravity feed was adjusted by changing the height difference between the inlet and exit reservoirs. Two 1-mL syringes were also connected to the microfluidic system for sample injection. The entire microfluidic system consisted of a polycarbonate flow introduction base allowing for inlet, outlet, and injection tubing connections, a poly(dimethylsiloxane) (PDMS)22 channel network adhered to the base, and a glass coverslip bonded to enclose the PDMS fluidic network. The PDMS fluidic network design used in these experiments is shown in Figure 1. The design incorporates a linear channel with two smaller injection channels near where the laser enters the flow cell. This design accommodates the laser and incorporates a channel several millimeters in length for separations and two smaller fluidic channels for spatially separated sample injections. The injection ports are located near the turn in the flow channel such that an injection is introduced near or below the focal point of the laser. This method of sample injection allows for a sample to be introduced to the OC beam in a more direct manner rather than relying on upstream injections. The resulting trapped particles are directed into the clean, particulate-free, flow above the focal point and are able to reside in the beam for an extended period of time undisturbed. To collect retention distance data for spores, pure injections were made and the resulting distances from the focal point recorded. Simultaneous separations were achieved through sequential injection of each spore type from the spatially separated injection channels. Strains. The following Bacillus strains were used in this study: B. anthracis Sterne strain 34F2 (nonpathogenic, vaccine strain) obtained from Colorado Serum Co. (Denver, CO) and B. (22) McDonald, J. C.; Whitesides, G. M. Acc. Chem. Res. 2002, 35, 491-499.

thuringiensis serovar. kurstaki strain 4D7 obtained from Bacillus Genetic Stock Center at The Ohio State University (Columbus, OH). Sporulation and Spore Purification. The overnight cultures of B. anthracis and B. thuringiensis were grown on trypticase soy agar plates (Difco, BD, Franklin Lakes, NJ) at 37 °C. A few colonies of each strain were resuspended in PBS buffer pH 7.0 and plated on 2×SG23 or NSM24 sporulation agar plates followed by incubation at 37 °C. The spores were collected as soon as the culture reached over 95% of phase bright spores, usually after 4 days, and resuspended in 2 mL of cold sterile MilliQ water. The suspension was centrifuged at 4000g for 5 min at 4 °C, and the resulting spore pellet was resuspended in a new portion of sterile MilliQ water. The spores were washed in this way four more times to remove the remaining debris and vegetative cells. B. anthracis spores were also obtained from liquid 2×SG medium. Briefly, 200 µL of overnight culture in trypticase soy broth (Difco, BD) was inoculated to 500 mL of 2×SG liquid sporulation medium. The culture was incubated with shaking at 30 °C for 4 days. The cultures were then centrifuged at 4000g for 15 min at 4 °C, and the resulting pellets were washed using the cold MilliQ water as in the case of solid medium sporulations. Sufficiently pure samples of B.t. grown in liquid 2×SG media could not be produced and so were not tested. Pure spore preparations were stored suspended in sterile MilliQ water at 4 °C. Spore Characterization. Spore size and morphology were characterized using transmission electron microscopy (TEM) with a JEOL 2200FS 200 kV field-emission microscope, with an in-column energy-loss filter (JEOL USA, Inc., Peabody, MA). The samples were prepared by pipetting spore suspensions onto holey carbon-film TEM supports, without use of stains or contrast agents. The surface tension of the solution forced the particles to maximize surface contact area on the TEM grid as it dried, and electrostatic forces ensured that no subsequent rotation occurred, so that the longest axes of the particles could be observed. The TEM images were recorded in zero-loss-filtered, bright-field imaging mode on a Gatan Ultrascan CCD (Gatan, Inc., Pleasanton, CA). To obtain accurate spore dimensions, the lengths and widths of at least 40 of each spore species were measured on the digital images. Refractive index was determined using a variation of the immersion method.25 Briefly, spores were suspended in liquids of varying refractive index (Immersion liquids series 5040, Cargille Laboratories, Cedar Grove, NJ) until they were indiscernible from the surrounding media. The refractive index of the liquid that resulted in their “disappearance” indicated the effective refractive index of the spores. The end point transparency was determined by measuring the percent transmission as a function of refractive index.26 RESULTS AND DISCUSSION The discovery of a significant optical chromatographic separation between two such closely related species of Bacillus spores opens new possibilities for the characterization and future separation of biological particles. The optical chromatographic difference (23) (24) (25) (26)

Leighton, T. J.; Doi, R. H. J. Biol. Chem. 1971, 246, 3189-3195. Phillips, A. P.; Ezzell, J. W. J. Appl. Bacteriol. 1989, 66, 419-432. Barer, R.; Ross, K. F. A.; Tkaczyk, S. Nature 1953, 171, 720-724. Hart, S. J.; Leski, T. A. Naval Research Laboratory Letter Report; 2005; Serial No.6110/255.

Figure 2. Images of (A) B. anthracis and (B) B. thuringiensis spores optically retained individually, and (C) optically retained simultaneously. The liquid flow was from right to left and the laser was propagating from left to right. Bright spots are due to laser light scatter from the spore (black rings used to highlight position). The laser focal point was positioned in the center of the main channel, 206 µm to the right of the inlet channel edge, seen in the upper left corner of each image. The scale bar represents 100 µm.

between the two related Bacillus spores studied in this correspondence can be seen in Figure 2, which shows images of the flow cell and laser light scatter from a B.a. spore (A), a B.t. spore (B) optically retained individually, and each spore type retained simultaneously (C). The identity of each spore in Figure 2C can be easily ascertained by noting the position of the spores measured individually, the B.a. spore being retained farther from the focal point than B.t. For each spore measurement, image data were collected every 5 s for 5 min (60 data points). Representative Z data from B.a. and B.t. separated in the optical chromatography system are shown in Figure 3. In this example the B.a. spore was retained at Z ) 1376 ( 9 µm and the B.t. spore retained at Z ) 587 ( 12 µm (∆Z ) 789 µm). The overall separation of these Bacillus spore species was greater than variations observed for the three different growth media employed in this study. Mean retention distance data for B.a. grown using liquid 2×SG, solid 2×SG, and solid NSM media were Z ) 1295 ( 109 µm (N ) 22), Z ) 1228 ( 138 µm (N ) 7), and Z ) 1207 ( 231 µm (N ) 11), respectively. By comparison, B.t. grown using solid 2×SG and NSM media had mean retention distances of Z ) 558 ( 86 µm (N ) 14) and Z ) 653 ( 158 µm (N ) 9). The B.a. samples were all retained above Z ) 1200 µm, and both B.t. sample types were retained at or below 653 µm. That B.a. spores are found at a greater retention distance than Analytical Chemistry, Vol. 78, No. 9, May 1, 2006

3223

Figure 3. Retention distance time lapse data for individually injected B.anthracis and B. thuringiensis spores.

are B.t. spores is consistent with B.a. experiencing a greater net force given the same fluid linear velocity (35 ( 1 µm/s) and laser power (1109 ( 26 mW) used in all experiments. The greater retention distance is due to either a larger optical force or smaller fluidic drag force acting on B.a. versus B.t. To further understand this phenomenon, each spore type was characterized with respect to refractive index, size/shape, and morphology. Refractive index studies of the spores revealed only small differences as measured in bulk suspension, B.a. n ) 1.532 and B.t. n ) 1.528, which can account for only a negligible fraction of the separation distance observed.15 However, differences in localized biological structures such as the spore coat may effect the apparent refractive index, not discernible in the bulk refractive index measurement.27 A larger apparent refractive index for B.a. could explain some of the large difference observed. The widths of the spores were similar, 0.71 ( 0.07 and 0.75 ( 0.07 µm for B.a. (N ) 41) and B.t. (N ) 40), respectively; the corresponding lengths were 1.28 ( 0.18 and 1.72 ( 0.20 µm for B.a. and B.t., respectively. It is well established that rod-shaped particles orient their length axially in the laser beam,28 and so the fluid drag force should be exerted continuously on the long axis of the spore.27 An estimate of the oblate spheroid fluid drag force27 calculated for B.t. (0.38 pN) is greater than that of B.a. (0.29 pN), which accounts for part of the ∼665-µm mean separation distance observed. Differences in the nature of the exosporium for each spore type were initially observed in our laboratory as “shadows” around the highly refractive spores using phase contrast light microscopy.29 The exosporium is a loose outer protective layer surrounding the much denser spore proper; it is present in certain spore species (e.g., B.a. and B.t.) and absent in others. TEM images of the B.t. spores seen in Figure 4, A and B, exhibit large regions of exosporia (light, less dense regions) extending out from each end of the B.t. spores (dark, denser regions). The exosporia present for both examples of B.a. in Figure 4, C and D, are much smaller and more evenly distributed (27) Kaneta, T.; Makihara, J.; Imasaka, T. Anal. Chem. 2001, 73, 5791-5795. (28) Gauthier, R. C.; Ashman, M.; Grover, C. P. Appl. Opt. 1999, 38, 48614869. (29) Gerhardt, P.; Ribi, E. J. Bacteriol. 1964, 88, 1774-&.

3224 Analytical Chemistry, Vol. 78, No. 9, May 1, 2006

Figure 4. Transmission electron microscope images of B. thuringiensis (A, B) and B. anthracis (C, D). The imaged spores were supported on holey carbon-film TEM supports seen as the gray webbed network behind the spores. Scale bars are 0.5 µm for (A) and (C) and 0.2 µm for (B) and (D).

around the spore than those seen for B.t. While both spore types possess an exosporium, the presence of a larger and more pronounced one in B.t. versus B.a. should increase the drag associated with B.t., further limiting its retention distance. The fact that the exosporia appear faint in phase contrast optical microscopy indicates their low refractive index relative to the spore proper. This is consistent with exosporium material not contributing significantly to the optical pressure and thus mainly affecting the fluidic drag force. CONCLUSION A significant retention distance difference (665-µm mean separation) between spores of B. anthracis and B. thuringinsis has been observed using optical chromatography. Morphologically, the less well retained B.t. spores were longer and had a much more pronounced exosporium compared with the B.a. spores. It is likely that the larger exosporium results in increased fluid drag force with little increase in its optical pressure size projection (due to low refractive index relative to the spore proper). Possible differences in the refractive index of the spore coat may increase the apparent refractive index of the spore while not influencing the overall refractive index measured in bulk.27 This localized refractive index effect may contribute to the retention distance difference between the spores. Additional work is underway to further understand this exciting discovery and the specific contributions that size, morphology, and possibly other variables have on the observed optical chromatography separations. The immediate potential for optical pressure-based separation/detection of the biological warfare agent, B. anthracis, based upon such differences is important for both military and civilian defense applications. In general, the existence of such significant differences observable in an optical pressure-based system highlights

the potential for finding other biological applications that exhibit similar traits.

for funding this research. Additional support was obtained through the Joint Service Agent Water Monitoring (JSAWM) program.

ACKNOWLEDGMENT The authors acknowledge the Office of Naval Research and the Naval Research Laboratory for support of this research. We also acknowledge the Defense Threat Reduction Agency (DTRA)

Received for review December 15, 2005. Accepted March 7, 2006. AC052221Z

Analytical Chemistry, Vol. 78, No. 9, May 1, 2006

3225