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Discrimination between Bacillus Species by Impedance Analysis of Individual Dielectrophoretically Positioned Spores Joseph D. Beck, Lu Shang, Bo Li, Matthew S. Marcus, and Robert J. Hamers* Department of Chemistry, University of WisconsinsMadison, 1101 University Avenue, Madison, Wisconsin 53706 We combine the use of dielectrophoretic positioning with electrical impedance measurements to detect and discriminate between individual bacterial spores on the basis of their electrical response. Using lithographically defined microelectrodes, we use dielectrophoresis to manipulate individual bacterial spores between the electrodes. The introduction of a single spore between the microelectrodes produces a significant change in electrical response that is species-dependent. When positioned between two electrodes and an AC voltage was applied, single spores caused current increases averaging 6.8 ((2.4) pA for Bacillus mycoides to 1.18 ((0.37) pA for Bacillus licheniformis. Using a mixture of spores of two different species, we demonstrate the ability to distinguish the species of individual spores in real time. This work demonstrates the feasibility of using impedance measurements for real-time detection and discrimination between different types of spores.
methods for detection of bacteria in vegetative and sporulated forms.10–12 One approach to electrical characterization of bacterial cells is the use of electrical impedance measurements to detect the ionic metabolites produced by bacterial growth.13–18 However, because dormant cells are not actively generating appreciable levels of metabolites, this approach is not readily applied to spores. A more direct approach to electrical detection has been a modification of the well-known Coulter counter in which a particle passing through an orifice separating two electrodes induces a change in current.19,20 While successfully applied to large cells such as red blood cells and yeast cells,19–21 much less work has been done on bacterial cells, which are typically much smaller and more difficult to detect. Electrical signals can also be used to actively separate and concentrate cells on the basis of their dielectric properties using methods such as dielectrophoresis.22–27 Recent studies using cells of Listeria28 and Escherichia coli10 have demonstrated the ability to separate viable from nonviable cells on the basis of differences in dielectrophoretic response, and in some cases, live cells of
The use of electrical signals to manipulate, to characterize, and to identify bacteria at the single-cell level remains a frontier area of analytical chemistry. Traditional biochemical methods such as genetic analysis,1–5 antibody recognition,4–7 and classic microbiological techniques7–9 are very effective at identifying bacteria. However, these are not easily adapted to rapid, real-time detection of individual cells. Recently, there has been increased interest in the use of all-electrical methods for detection and analysis at the single-cell level and especially in the development of improved
(10) Lapizco-Encinas, B. H.; Simmons, B. A.; Cummings, E. B.; Fintschenko, Y. Anal. Chem. 2004, 76, 1571. (11) Liu, Y. S.; Walter, T. M.; Chang, W. J.; Lim, K. S.; Yang, L. J.; Lee, S. W.; Aronson, A.; Bashir, R. Lab Chip 2007, 7, 603. (12) Popovtzer, R.; Natan, A.; Shacham-Diamand, Y. J. Electroanal. Chem. 2007, 602, 17. (13) Firstenberg-Eden, R.; Eden, G. Impedance Microbiology; Wiley Press: New York, 1984. (14) Silley, P.; Mortimer, F. In Rapid Microbiological Methods in the Pharmaceutical Industry; Easter, M. C., Ed.; CRC Press: Boca Raton, Florida, 2003; p 99. (15) Noble, P. A.; Dziuba, M.; Harrison, D. J.; Albritton, W. L. J. Microbiol. Methods 1999, 37, 51. (16) Wawerla, M.; Stolle, A.; Schalch, B.; Eisgruber, H. J. Food Prot. 1999, 62, 1488. (17) Sheppard, N. F.; Tucker, R. C.; Wu, C. Anal. Chem. 1993, 65, 1199. (18) Gomez, R.; Bashir, R.; Bhunia, A. K. Sens. Actuators, B: Chem. 2002, 86, 198. (19) Sohn, L. L.; Saleh, O. A.; Beavis, A. J.; Allan, R. S.; Notterman, D. A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10687. (20) Cheung, K.; Gawad, S.; Renaud, P. Cytometry Part A 2005, 65A, 124. (21) Vilkner, T.; Janasek, D.; Manz, A. Anal. Chem. 2004, 76, 3373. (22) Pysher, M. D.; Hayes, M. A. Anal. Chem. 2007, 79, 4552. (23) Voldman, J.; Gray, M. L.; Toner, M.; Schmidt, M. A. Anal. Chem. 2002, 74, 3984. (24) Li, H. B.; Zheng, Y. N.; Akin, D.; Bashir, R. J. Microelectromech. Syst. 2005, 14, 103. (25) Pethig, R.; Markx, G. H. Trends Biotechnol. 1997, 15, 426. (26) Fiedler, S.; Shirley, S. G.; Schnelle, T.; Fuhr, G. Anal. Chem. 1998, 70, 1909. (27) Hughes, M. P. Electrophoresis 2002, 23, 2569. (28) Li, H.; Bashir, R. Sens. Actuators, B 2002, 86, 215.
* To whom correspondence should be addressed. E-mail:
[email protected]. (1) Shepard, J. R. E.; Danin-Poleg, Y.; Kashi, Y.; Walt, D. R. Anal. Chem. 2005, 77, 319. (2) Koh, C. G.; Tan, W.; Zhao, M. Q.; Ricco, A. J.; Fan, Z. H. Anal. Chem. 2003, 75, 4591. (3) Gasanov, U.; Hughes, D.; Hansboro, P. M. FEMS Microbiol. Rev. 2005, 29, 851. (4) Bettelheim, K. A.; Beutin, L. J. Appl. Microbiol. 2003, 95, 205. (5) Maciorowski, K.; Pillai, S.; Ricke, S. J. Appl. Microbiol. 2000, 89, 710. (6) Rowe, C. A.; Tender, L. M.; Feldstein, M. J.; Golden, J. P.; Scruggs, S. B.; MacCraith, B. D.; Cras, J. J.; Ligler, F. S. Anal. Chem. 1999, 71, 3846. (7) Al Dahouk, S.; Tomaso, H.; Nockler, K.; Neubauer, H.; Frangoulidis, D. Clin. Lab. 2003, 49, 487. (8) Rompre, A.; Servais, P.; Baudart, J.; de-Roubin, M. R.; Laurent, P. J. Microbiol. Methods 2002, 49, 31. (9) Maciorowski, K.; Herrera, P.; Jones, F.; Pillai, S.; Ricke, S. Vet. Res. Commun. 2006, 30, 127. 10.1021/ac702113t CCC: $40.75 2008 American Chemical Society Published on Web 04/11/2008
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different species have been separated.29 In these studies, however, the actual detection of the separated cells was still performed optically. More recently, Suehiro and co-workers30,31 have combined dielectrophoresis with electrical impedance measurements to manipulate and characterize the response of pearl-chains of bacteria spanning interdigitated electrodes. We recently showed that dielectrophoretic manipulation could be combined with impedance-based electrical measurement to capture, to transport, and to detect individual bacterial cells (such as Bacillus mycoides) and other nanoscale objects by using pairs of microfabricated electrodes separated by a small gap roughly the size of a single bacterial cell and by using nulling techniques to reduce the influence of stray capacitance of the aqueous medium.32,33 The use of dielectrophoresis is important because it provides a way to transport individual cells into the analytical measurement area, to measure their electrical response, and then to release them; in this way, large numbers of individual cells can be characterized sequentially with a single set of electrodes. While our previous work demonstrated the ability to characterize individual bacterial cells in their vegetative state, there is great interest in the ability to characterize and to identify bacterial spores,34,35 such as Bacillus anthracis. Many species of Bacillus are genetically similar and have similar proteins exposed on the outer surface of the spore coat making analysis in the sporulated state more challenging than in the vegetative state.36,37 Indeed, most approaches to analysis of spores rely on either digestion36 or germination to the vegetative state, which is time-consuming and cumbersome.38 Here, we demonstrate the ability to rapidly detect individual bacterial spores via electrical measurements. We show that the magnitude of the electrical response when an individual spore enters a gap between two planar microelectrodes can be used to discriminate between different Bacillus species and in some cases can even achieve discrimination at the subspecies level. Measurement of the electrical response of individual spores provides a way to discriminate between different bacterial spores that can be used in conjunction with additional separation and identification methods as part of an integrated microfluidic analysis system.39 The use of planar micrometer-sized electrodes can easily be scaled up to large numbers of electrodes for highly parallel detection and separation at the single-cell level. (29) Markx, G. H.; Dyda, P. A.; Pethig, R. J. Biotechnol. 1996, 51, 175. (30) Suehiro, J.; Yatsunami, R.; R., H.; Hara, M. J. Phys. D: Appl. Phys. 1999, 32, 2814. (31) Suehiro, J.; Noutomi, D.; Shutou, M.; Hara, M. J. Electrost. 2003, 58, 229. (32) Beck, J. D.; Shang, L.; Marcus, M. S.; Hamers, R. J. Nano Lett. 2005, 5, 777. (33) Marcus, M. S.; Shang, L.; Li, B.; Streifer, J. A.; Beck, J. D.; Perkins, E.; Eriksson, M. A.; Hamers, R. J. Small 2007, 3, 1610. (34) Ivnitski, D.; Abdel-Hamid, I.; Atanasov, P.; Wilkins, E. Biosens. Bioelectron. 1999, 14, 599. (35) Lazcka, O.; Del Campo, F. J.; Munoz, F. X. Biosens. Bioelectron. 2007, 22, 1205. (36) Swatkoski, S.; Russell, S. C.; Edwards, N.; Fenselau, C. Anal. Chem. 2006, 78, 181. (37) Swiecki, M. K.; Lisanby, M. W.; Shu, F. Y.; Turnbough, C. L.; Kearney, J. F. J. Immunol. 2006, 176, 6076. (38) Williams, D. D.; Benedek, O.; Turnbough, C. L. Appl. Environ. Microbiol. 2003, 69, 6288. (39) Breadmore, M. C.; Wolfe, K. A.; Arcibal, I. G.; Leung, W. K.; Dickson, D.; Giordano, B. C.; Power, M. E.; Ferrance, J. P.; Feldman, S. H.; Norris, P. M.; Landers, J. P. Anal. Chem. 2003, 75, 1880.
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Figure 1. Diagram of electrical apparatus for dielectrophoretic manipulation and detection. The image shows four electrodes. Left and right electrodes are separated by ∼3 µm. Both left (excitation) electrodes have been connected to the output from the internal oscillator output of the dual-phase lock-in amplifier. The receiving electrode at right is connected to the input of the dual-phase lock-in amplifier. The electrode at top right can be connected to the receiving electrode or can be left disconnected (floating).
EXPERIMENTAL SECTION Microchip and Fluidic Chamber. Our assembly consisted of a polyacrylic base, a micropatterned chip, a polydimethylsiloxane (PDMS) mold with hollow chamber, and a glass coverslip. Gold electrodes were fabricated onto quartz wafers using standard optical lithography and evaporation techniques. A 3 × 2 × 1.5 mm chamber was fabricated in the PDMS mold with microfluidic channels for fluid flow. The bottom of the chamber was directly exposed to the micropatterned chip, while the top was exposed to a glass coverslip. This arrangement permitted the spores to be directly observed and to be recorded on a microscope-mounted video camera during electrical manipulation and measurement thereby directly confirming that nature of the electrical response. Electrical Apparatus. Two pairs of electrodes were patterned on each chip. Each patterned electrode was electrically connected to a BNC connection through a Signatone probe (model DS345). Electrical excitation signals were applied to two electrodes; one of these was directly connected to the internal oscillator output of a dual-phase lock-in amplifier (Signal Recovery model no. 7265). The second excitation electrode was connected to a custom-made inverter circuit that took the same signal from the oscillator and that shifted its phase by 180°. Two “receiver” electrodes were fabricated directly opposite the two excitation electrodes separated by a gap of 3.5 µm. Current flowing through the receiver electrodes was amplified using a Femto model DLPCA-200 transimpedance amplifier (current-to-voltage converter). In principle, connecting the two receiver electrodes together at the low-impedance input to the current amplifier forms a bridge circuit in which the capacitive currents in the two receiver electrodes are inverted replicas of one another such that only the difference in current between the two receiver electrodes is amplified.33 In practice, we found that because the input noise of the current-to-voltage converters depends on the input capacitance, better results were obtained leaving one “receiving” electrode floating and connecting only the “measurement” electrode to the current amplifier as depicted in Figure 1. This arrangement leads to slightly imperfect nulling (∼90% rejection of common-mode signals) because of the difference in distance to the two excitation electrodes but leads to lower output noise. Using
either cancellation scheme, we were able to detect the changes in impedance when a single bacterial spore entered the gap between the excitation electrode and the opposing measurement electrode. Spore Preparation. Bacillus cereus ATCC no. 14579 was obtained from Microbiologics (St. Cloud, MN). All other Bacillus strains were obtained from Bacillus Genetic Stock Center. These included B. mycoides (BGSC no. 6A11), Bacillus subtilis (BGSC no. 1E7), Bacillus licheniformis (BGSC no. 5A36), and B. licheniformis (BGSC no. 5A24). Bacillus spores were generated by plating vegetative cells onto a 10 cm plate of Schaeffer’s sporulation agar. After 7 days at room temperature, the cell mass was scraped off the agar with a glass rod and then was rinsed with deionized water into a centrifuge tube. After centrifugation (3000g) for 5 min, the water was removed leaving behind a spore pellet. The pellet was rinsed three more times in water and was resuspended a final time in 2 mL of deionized water and was stored at 4 °C until use. Bright-field microscopic analysis showed uniformly sized, phase-bright objects that resisted staining by safranin. The inability to stain with safranin confirms that the cells were in a spore form. A 60 °C heat treatment followed by dilution plating onto nutrient agar showed that each 10 cm spore plate generated at least 100 million colony-forming units. Spore suspensions were diluted at 1:200 in deionized water for impedance analysis. The species investigated are all categorized as biosafety level one. Solid biological waste was autoclaved. Liquid biological waste was treated with Clorox bleach solution (diluted ∼1:4 in water) for a minimum of 1 hour. RESULTS AND DISCUSSION Dielectrophoretic Positioning of Single Spores. The spores exhibited dielectrophoretic movement similar to what we reported previously using vegetative cells of B. mycoides.32 Application of an AC voltage to the electrodes produces an electric field that is high at the electrode edges and that is highest in the gap between electrodes. The electric field pulls spores from the bulk to the electrode edge. Each loosely held spore is then propelled by fluid flow until it reaches the end of the electrode partially spanning the gap between the excitation and the receiver electrodes (Figure 2). Videos showing this process for vegetative B. mycoides cells were published previously.32 Figure 2 shows selected still images for spores of B. mycoides. The capture and positioning of individual spores was successful at frequencies between 10 and 100 kHz. As expected, the amplitude of the applied excitation voltage greatly affected capture efficiency and spore positioning. A root-meansquare (rms) amplitude of 10 spores from each species.
Figure 4. Changes in current induced by individual B. subtilis spores measured at different frequencies. The average current increase is plotted. Each point represents the average of >15 measurements of different cells at each frequency. Error bars represent 1 standard deviation.
These spore-dependent increases in current were dependent on the frequency of the applied signal. As shown in Figure 4, single B. subtilis spores caused an average 1.3 ± 0.5 pA change in current at 10 kHz and 30 kHz, but at 100 kHz, the average increase was 5 ± 2.3 pA (Figure 4). Measurements below 50 kHz were obtained using a gain of 107 V/A, but at 75 kHz and 100 kHz, the necessary amplifier bandwidth could only be obtained at a lower gain setting of 106 V/A. Although both gain settings generated similar results for data acquired at 30 kHz (data not shown), the 106 setting yields a poorer signal-to-noise ratio that is reflected in a larger spread of values at the higher frequencies. Despite this increased variation, the current increases measured at 75 kHz and 100 kHz are significantly larger than those measured at 30 kHz with a confidence level of >99% (Student’s t-test). As data were acquired from only a few frequencies spanning a relatively short range, we cannot make firm conclusions regarding the mechanism of conduction. However, our data do appear largely consistent with previous studies that analyzed the electrical properties of spore suspensions. Carstensen et al.40 showed that the electrical properties of Bacillus spores can be represented as multiple concentric shells that alternate between relatively low impedance regions (core, cortex, spore coat) and high impedance lipid regions (plasma membrane, outer membrane, exosporium). AC fields at frequencies below ∼100 kHz are not expected to conduct appreciable current across lipid membranes.40 Consequently, we believe that the observed increases in current are due to surface charges exterior to lipid membranes. The fixed charges on the spore surface are surrounded by an (40) Carstensen, E. L.; Marquis, R. E.; Child, S. Z.; Bender, G. R. J. Bacteriol. 1979, 140, 917.
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Figure 6. Histogram demonstrating the ability to achieve real-time discrimination of individual spores within a mixture. A mixture of B. mycoides and B. subtilis spores was flowed over the electrodes, and individual spores were captured and measured. Simultaneous microscopy observations permitted visual identification of the species being measured. The histogram shows the number of spores measured that fell within a given range of current change. The hatched bars represent B. subtilis. The white bars represent B. mycoides.
electrical double layer. It is likely that the increase in current between 30 and 75 kHz is associated with this layer, but more experiments will be required to definitively establish the underlying mechanisms. Electrical Discrimination of Species. To test the ability to distinguish between different species on the basis of their electrical response, we measured multiple species of Bacillus spores. Measurements shown here were performed at 10 kHz. Figure 5 summarizes the changes in current observed for five different Bacillus species and subspecies. The increases in current for this set of experiments ranged from an average of 6.8 ± 2.4 pA for B. mycoides (n ) 11) to 1.18 ± 0.37 for B. licheniformis 5A36 (n ) 17). Although B. mycoides produces a much larger spore, the differences cannot be due merely to cell volume. This is evident as B. cereus and B. licheniformis produce spores of similar size, and yet B. cereus creates a current increase 4 times that of B. licheniformis 5A36. The signals observed here for spores (∼1-7 pA) are significantly smaller than the values of ∼40 pA reported previously for vegetative cells using a similar electrode geometry.32
To further confirm that the results in Figure 5 reflect speciesspecific differences in the spore and not other factors such as charged impurities in the solvent (as might be released from the mostly dormant spores), we conducted experiments using mixtures of cell species. Figure 6 shows a histogram of the changes in electrical current observed using a mixture of B. mycoides and B. subtilis spores. B. mycoides was chosen because its spore is larger than the other species thereby permitting us to directly confirm the identity of the spore being measured using optical microscopy simultaneously with the electrical measurements. B. subtilis was chosen because this species caused a significantly smaller current change than B. mycoides yet would be a more challenging test of the system than choosing B. licheniformis 5A36, which had the largest disparity with B. mycoides. In this experiment, all 38 B. subtilis spores caused current increases ranging between 0.6 and 1.6 pA. In contrast, all 34 B. mycoides spores caused increases ranging from 1.6 to 4.2 pA. Thus, the electrical data provide essentially perfect discrimination between these two species using a threshold of 1.6 pA. Different bacterial species have long been known to vary in their amino acid, saccharide, and dipicolinic acid compositions.41 It is therefore not surprising that surface hydrophilicity varies dramatically between species as well.42,43 The surface charges that confer hydrophilicity to spores may serve as charge carriers in the presence of an electrical potential. Species-specific variations in the abundance and character of surface charge carriers may explain the species-specific variations seen in both hydrophobicity and impedance. (41) Warth, A. D.; Ohye, D. F.; Murrel, W. G. J. Cell Biol. 1963, 16, 579. (42) Koshikawa, T.; Yamazaki, M.; Yoshimi, M.; Ogawa, S.; Yamada, A.; Watabe, K.; Torii, M. J. Gen. Microbiol. 1989, 135, 2717. (43) Wiencek, K. M.; Klapes, N. A.; Foegeding, P. M. Appl. Environ. Microbiol. 1990, 56, 2600.
CONCLUSIONS The above results represent the first demonstration of single-spore, impedance-based discrimination between closely related bacterial species. The ability to capture individual spores and to release them at will provides a flexible way to interrogate the electronic properties of individual spores for separation and possible identification. Since spores of a particular species of interest give rise to an electrical response lying within a welldefined confidence interval, measurement of the response of individual cells in a more complex sample could provide a firstpass discrimination in an integrated detection system in which “outliers” that yield responses outside of the confidence interval would be attributed to different species or other sources (e.g., particulates or contamination). The use of electrical impedance measurements to identify or categorize individual spores may be particularly useful in conjunction with other physical or chemical discrimination methods as one element of an integrated real-time biological detection system. While here we use a single sinusoidal waveform both to capture and to interrogate the properties of the captured spores, it is possible to sum waveforms of different frequencies and amplitudes to separate these functions and therefore to perform more sophisticated electrical analyses.32 ACKNOWLEDGMENT This work is supported in part by the National Science Foundation Grant DMR-0420885 and by Smiths Detection, Inc.
Received for review October 15, 2007. Accepted March 9, 2008. AC702113T
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