Soil Martian Biosensors Based on Dielectric Spectroscopy
by John H. Miller, Jr., and David Warmflash
candidate for the ABSTRACT--Researchers are studying the elecpossible existence tromagnetic responses of live organisms, and the of life, either in the distant potential of such measurements to develop biosenpast or at present,1 for many sors with applications in astrobiology and medidecades. If life were to be discine. For example, dielectric spectroscopy meascovered on Mars, the scientifurements at different temperatures can distinguish ic implications would be prolive organisms from nonliving complex macromolfound for the distribution of ecules and may eventually be suitable for in situ life in the cosmos and the evoastrobiology studies on the surface of Mars or in lution of life on Earth. In the liquid ocean beneath the ice of Europa. More 1976, the Viking program recent studies have involved nonlinear (harmonic made an attempt to detect evigeneration) responses of biological systems to oscildence for living or fossilized latory electric fields. Some results suggest that organisms in Martian soil, active biological motors and other enzyme comwhich yielded ambiguous, plexes generate harmonics over specific frequency somewhat negative results.2 ranges. These include complexes in the mitochonThe exciting, and more recent, drial inner membrane, such as the molecular turstudies3 of the Martian metebine ATP synthase and pumps in the outer plasma orite Allan Hills 84001 John H. Miller, Jr. membrane. In addition, the harmonic generation (ALH84001) suggest that spectra of chloroplasts, responsible for photosynmicrobial life existed on Mars thesis in plants, exhibit light-activated features. This provides evidence that the techabout four billion years ago. nique detects physiologically active processes, which could lead to fundamental Reported evidence includes advances in understanding of biochemical and other complex macromolecular systems. magnetite (Fe3O4) crystals found in carbonate globules and their associated rims in the meteorite.4 About one fourth of on frequency and on each enzyme's charge distribution, structure, these tens-of-nanometer sized magnetites are nearly identical to and state of activity. The resulting motion of charged macromolthose produced by magnetotactic bacteria on Earth and are not ecules leads to a nonlinear response and to the generation of highexpected to be produced by abiotic means. It has thus been argued er harmonics, providing a powerful functional spectroscopy tool. that such Martian magnetite crystals are magnetofossils, which, if This hypothesis is supported by the following observations. true, would constitute evidence of the oldest life forms known.5 First, oscillatory fields induce ac components of transmemFurther studies indicate that subsurface Martian life could brane potentials that add to the intrinsic potentials.12 A low-frepotentially survive even today.6 There is geological evidence that quency electric field polarizes live cells or macromolecules,13 ice was once deposited in the regolith, where it may be present resulting in enormous dielectric responses, and also modulates above mid-latitudes.7 This ice, which could extend several kilome- the membrane potential of each cell.14 Second, sinusoidal fields ters below the surface, might be a source of liquid water near mag- can induce membrane pumps to translocate cations15,16 and genmatic intrusions.8 On Earth, the biomass of subterranean organ- erate harmonics.17 Membrane proteins exhibit nonlinear behavisms may even exceed that at the surface.9 These organisms can ior18 since domains with dipole moments interact with the live in highly saline conditions at temperatures from 115C to induced transmembrane potential, driving them to change con20C.10,11 Such conditions might prevail beneath the surface in an formation. The combination of conformational changes and ion aquifer or hydrothermal system. Therefore, there is considerable translocation creates a nonlinear response. For example, cation interest in developing new techniques for detecting subsurface life pumps such as P-type ATPases,19 have been reported to generon Mars. Furthermore, the likelihood that oceans of liquid water ate harmonics.20 We have developed a sensitive method,21 using exist below the icy surfaces of Europa and other moons make superconducting quantum interference devices (SQUIDs) to these exciting candidates for possible extraterrestrial life. measure the harmonics produced by such membrane pumps at This research is further motivated by the hypothesis that an low frequencies. Further support is provided by our recent haroscillatory field induces proteins and other macromolecules to monic generation spectroscopy measurements, which will be change conformation. The rate of conformational change depends discussed in the section "Results and Discussion."
M
ARS HAS BEEN A
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Goal of the Project The goal of this project is to study dielectric spectroscopy22 and related methods, such as harmonic generation spectroscopy,23 for the detection of live organisms. Toward this end, the project aims to identify and explain possible signatures of active macromolecular complexes unique to living biological systems. Possible signatures include unusual behavior, distinct from those of inanimate materials, in the frequency- and temperature-dependent dielectric response, and in harmonic generation spectroscopy, in which generated harmonics are plotted vs. frequency and amplitude. Methodology Previous reports focused on variable temperature dielectric spectroscopy of live organisms and Martian soil simulants. Our experiments on linear dielectric response employed a Solartron Impedance Analyzer, which measures complex admittance at frequencies up to 32 MHz. In this report, we report on harmonic generation spectroscopy measurements that suggest a potentially unique method of detecting physiologically active processes. A four-electrode setup is employed in conjunction with a Stanford Research SR780 signal analyzer, operated as a spectrum analyzer, for measurements at kilohertz frequencies. A function generator applies a sinusoidal signal to the outer electrodes, while the voltage difference between the inner electrodes is fed into Channel 1 of the SR780, which records the induced harmonics. A reference spectrum is acquired using a supernatant, whose conductivity has been adjusted (with distilled water, to compensate for the volume fraction of the cells present in the sample) to be identical to that of the sample at the frequency of the point of interest. The supernatant typically consisted of an aqueous solution of ~1- 10 mM NaCl. Two different types of control files are used, depending upon whether the reference is to be logged using the same set of electrodes or a separate matched reference cell. In either case, the logging, windowing, and Fourier Transform routines were identical and provide a power spectrum of the reference cell, which is also recorded as a data file in the computer. Finally, the sample power spectrum obtained from the sample (e.g., cell suspension or soil sample) of interest is divided by the reference power spectrum, and also stored. The entire procedure is automated using LabVIEW data acquisition software. The power of this approach stems from the fact that it allows the researcher to deconvolve the effects of nonlinearities within the electrochemical system from those attributed to the biological cells themselves. Equipment and Special Technology Most of our experiments at kilohertz frequencies employ the 4electrode configuration shown in Fig. 1, where electrodes are immersed into a suspension of cells or organelles. In Fig. 1, the waveform generator applies a sinusoidal voltage of high spectral purity to the two outer electrodes, while the response across the inner electrodes is measured with a Stanford Research SR780, which shows the generated higher harmonics. Typically, the second or third harmonic generated by the suspension is recorded vs. amplitude and frequency, and all measurements are automated with LabVIEW software. Experiments have been carried out on suspensions of whole
Figure 1. Schematic diagram of 4-electrode setup for in vitro measurements, in which platinum electrodes are immersed directly into a suspension (expanded scale) of cells, organelles, or reconstituted vesicles. The spacing between the outer electrodes is about 1 cm and the housing rests on a magnetic stirrer. The Agilent 33220A Waveform Generator produces a sinusoidal voltage of high spectral purity, while the Stanford Research SR780 Dynamic Signal Analyzer is operated as an FFT (fast Fourier transform) spectrum analyzer. Both instruments are interfaced to a computer (not shown) using a GPIB (general purpose interface bus). The experiments are automated with LabVIEW software. The oxygen sensor enables one to monitor the rate of oxygen consumption (or production for the case of chloroplasts), which can be correlated with the generation of harmonics created by enzymatic activity.
cells, mitochondria, and chloroplasts, the latter of which provide validation and are of fundamental interest since the photosynthetic enzyme complexes are light activated. Many of these measurements have been carried out in collaboration with the group headed by Prof. William R. Widger in the Department of Biology and Biochemistry at the University of Houston. Additional experiments have been performed on whole organisms, such as plants and earthworms. Results and Discussion Some examples of observed harmonic generation spectra in the kHz range are illustrated by Fig. 2, which shows generated harmonics vs. applied frequency for S. cerevisiae, which lacks mitochondrial complex I, and uncoupled mammalian mitochondria, in which complexes I, III, and IV have been activated by glutamate malate but ATP synthase (complex V) is inactive due to the lack of a transmembrane potential. (We observed similar, though not identical, behavior by activating complexes II, III, and IV with succinate.) Several features of the harmonic spectra are noteworthy. First, the spectral features generated by S. cerevisiae increase with cell concentration (upper left). We also find that potassium cyanide, which binds to complex IV and completely shuts down ATP production, suppresses the observed spectral features, as seen in the B. indicas data shown here. Second, the higher frequency feature (~12 kHz) observed in S. cerevisiae and B. indicas is not seen in uncoupled mitochondria, in which
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Figure 2. Harmonic generation spectra (applied field amplitude = 5 V/cm) of suspensions of budding yeast (S. cerevisiae, top), a relative of the mitochondrial ancestor (B. indicas, lower left, and uncoupled mammalian mitochondria, in which ETC complexes I, III, and IV have been activated by adding glutamate malate (lower right). The data were obtained with the 4-electrode setup shown in Fig. 2, in which a sinusoidal voltage is applied across the outer two electrodes and the induced harmonics measured across the two inner electrodes with a spectrum analyzer. The spectral features generated by S. cerevisiae are observed to increase with cell concentration (upper left).
Figure 3. (Left) Generated second harmonic vs. fundamental frequency (applied field amplitude = 5 V/cm) of a suspension of uncoupled mammalian mitochondria, in which ETC complexes I, III, and IV are activated by adding glutamate malate (closed circles) and subsequently inhibited (open circles) by adding rotenone, which binds to complex I. Here the second harmonic was by far the largest of the higher harmonics, and the magnitude of the second harmonic response (2-40 kHz) was measured for applied frequencies ranging from 1 to 20 kHz. (Right) Generated second harmonic vs. fundamental frequency (field amplitude = 1 V/cm, 4-electrode method) of a suspension of uncoupled mammalian mitochondria before (open circles) and after (closed circles) activating ETC complexes II, III, and IV with succinate.
ATP (complex synthase V) is inactive. We hypothesize that the higher frequency feature is generated during ATP production by this molecular turbine. Finally, note that, although a lower frequency (~3 kHz) peak in harmonic generation spectra is present in S. cerevisiae, B. indicas, and uncoupled mitochondria, this feature is significantly smaller in S. cerevisiae. There could be at least two possible reasons for this. One is that complex I is missing in S. cerevisiae, and it may contribute to the more pronounced feature seen in B. indicas and mitochondria. This is consistent with data obtained from uncoupled mitochondria, Fig. 3, in which either complexes I, III, and IV or complexes II, III, and IV are activated. In addition, the plasma membrane of a eukarote, such as S. cerevisiae, acts as a high-pass filter due to its finite capacitance. The data in Fig. 3 was also obtained with the 4electrode method, in which a sinusoidal voltage is applied across
the outer two electrodes and the induced harmonics measured across the two inner electrodes with a spectrum analyzer. We find that potassium cyanide, which binds to complex IV and completely shuts down ATP production, suppresses the observed spectral features, as seen in the B. indicas data shown here. The higher frequency feature (~12 kHz) observed in whole cells is not seen in uncoupled mitochondria, where ATP synthase (complex V) is inactive. We hypothesize that the higher frequency feature is generated during ATP production by this molecular turbine. Photosynthetic organisms have also proven extremely useful for validation of the technique, since photosynthesis and ATP production are activated by light. Figure 4 shows examples of the second harmonic generation spectra of a whole leaf and a suspension of spinach chloroplasts, which show dramatic differences between the spectra with and without light activation.
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Figure 4. Light activated responses in the 2nd harmonic generation spectra of a whole leaf (left) and a suspension of spinach chloroplasts (right) in which the photosynthetic ETC has been activated by an electron acceptor (ferricyanide).
Conclusions Our studies thus far have encompassed both linear (impedance, dielectric) and nonlinear (harmonic generation, mixing) responses of biological systems to oscillatory electric fields. Some results suggest that active biological motors and other enzyme complexes generate harmonics over specific frequency ranges. These include complexes in the mitochondrial inner membrane, such as the molecular turbine ATP synthase, and pumps in the outer plasma membrane. In addition, the harmonic generation spectra of chloroplasts, responsible for photosynthesis in plants, exhibit light-activated features. This provides evidence that the technique detects physiologically active processes, which could lead to fundamental advances in understanding of biochemical and other complex macromolecular systems. Moreover, the method could eventually play an important role in the search for extant life elsewhere in the solar system. References 1 B. M. Jakosky and E. L. Shock, "The Biological Potential of Mars, the Early Earth, and Europa," J. Geophys. Res. 103 (1998): 19,359-19,364. 2 L. Margulis, P. Mazur, E. S. Barghoorn, H. O. Halvorson, T. H. Jukes, and I. R. Kaplan, "The Viking Mission: Implications for Life on Mars," J. Mol. Evol. 14 (1979): 223-32. 3 D. S. McKay, E. K. Gibson Jr., K. L. Thomas-Keprta, H. Vali, C. S. Romanek, S. J. Clemett, X. D. F. Chillier, C. R. Maechling, and R. N. Zare, "Search for Past Life on Mars: Possible Relic Biogenic Activity in Martian Meteorite ALH84001," Science 273 (1996): 924-930. 4 K. L. Thomas-Keprta, S. J. Clemett, D. A. Bazylinksi, J. L. Kirschvink, D. S. McKay, S. J. Wentworth, H. Vali, E. K. Gibson, Jr., M. F. McKay, and C. S. Romanek, "Truncated Hexaoctahedral Magnetite Crystals in ALH84001: Presumptive Biosignatures," Proc. Nat. Acad. Sci. USA 98 (2001): 2164-69. 5 K. L. Thomas-Keprta, S. J. Clemett, D. A. Bazylinksi, J. L. Kirschvink, D. S. McKay, S. J. Wentworth, H. Vali, E. K.
Gibson, Jr., and C. S. Romanek, "Magnetofossils from Ancient Mars: A Robust Biosigniture in the Martian Meteorite ALH84001," Applied & Environmental Microbiology 68 (2002): 3663-3672. 6 B. P. Weiss, Y. L. Yung, and K. H. Nealson, "Atmospheric Energy for Subsurface Life on Mars?" Proc. Nat. Acad. Sci. USA 97 (2000): 1395-1399. 7 M. T. Mellon and B. M. Jakosky, "Geographic Variations in the Thermal and Diffusive Stability of Ground Ice on Mars," J. Geophys. Res. 98 (1993): 3345-64. 8 M. H. Carr, Water on Mars. New York: Oxford Univ. P., 1996. 9 W. B. Whitman, D. C. Coleman, and W. J. Wiebe, "Prokaryotes: The Unseen Majority," Proc. Nat. Acad. Sci. USA 95 (1998): 6578-83. 10 K. H. Nealson, "The Limits of Life on Earth and Searching for Life on Mars," J. Geophys. Res. 102 (1997): 23,675-86. 11 J. C. Priscu, C. H. Fritsen, E. E. Adams, S. J. Giovannoni, H. W. Paerl, C. P. McKay, P. T. Doran, D. A. Gordon, B. D. Lanoil, and J. L. Pinckney, "Perennial Antarctic Lake Ice: An Oasis for Life in a Polar Desert," Science 280 (1998): 2095-98. 12 H. P. Schwan. "Electrical Properties of Tissues and Cells," Adv. Biol. Med.Phys. 5 (1957): 147-209. 13 H. Sanabria, J. H. Miller, Jr., A. Mershin, R. F. Luduena, A. A. Kolomenski, H. A. Schuessler, and D. V. Nanopoulos, "Impedance Spectroscopy of a-b Tubulin Heterodimer Suspensions," Biophys J. 90 (2006): 4644-50. 14 C. Grosse and H. P. Schwan. "Cellular Membrane Potentials Induced by Alternating Fields," Biophys J. 63 (1992): 1632-164. 15 R. D. Astumian, "Effects of Time-dependent Electric Fields on Membrane Transport," Biophys J. 64 (1993): 7-8. 16 R. D. Astumian and I. Dernyi, "Fluctuation Driven Transport and Models of Molecular Motors and Pumps," Eur. Biophys. J. 27 (1998): 474-89. 17 A. M. Woodward and D. B. Kell, "On the Nonlinear Dielectric Properties of Biological Systems. Saccharomyces cerevisiae," Bioelectrochem. Bioenerg. 24 (1990): 83-100.
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D. Nawarathna, J. R. Claycomb, J. H. Miller, Jr., and M. J. Benedik, "Nonlinear Sielectric Spectroscopy of Live Cells Using Superconducting Quantum Interference devices," Appl. Phys. Lett. 86 (2005): 023902-1-3.
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ELECTROMAGNETIC RESPONSES--Hans Infante, graduate student in physics, takes in-vitro measurements of electromagnetic responses of living cell suspensions utilizing superconducting quantum interference devices.
Publications Sanabria, Hugo, John H. Miller, Jr., Andreas Mershin, Richard F. Luduena, Alexandre A. Kolomenski, Hans A. Schuessler, and Dimitri V. Nanopoulos, "Impedance Spectroscopy of - Tubulin Heterodimer Suspensions," Biophysical Journal 90 (2006): 4644-4650. Nawarathna, D., J. R. Claycomb, G. Cardenas, J. Gardner, D. Warmflash, J. H. Miller, Jr., and W. R. Widger, "Harmonic Generation by Yeast Cells in Response to Low-Frequency Electric Fields," Physical Review E 73 (2006): 051914-1--6. Claycomb, James R. and John H. Miller, Jr., "Superconducting and High-Permeability Shields Modeled for Biomagnetism and Nondestructive Testing," IEEE Trans. on Magnetics 42 (2006): 1694-1702. Sanabria, Hugo and John H. Miller, Jr., "Relaxation Processes Due to the Electrode-Electrolyte Interface in Ionic Solutions," Physical Review E 74 (2006): 051505--1-9. Mershin, Andreas, Hugo Sanabria, John H. Miller, Dharmakeerthna Nawarathna, Efthemios M. C. Skoulakis, Nikolaos E. Mavromatos, Alexandre A. Kolomenskii, Hans A. Schuessler, Richard F. Luduena, and Dimitri V. Nanopoulos, "Towards Experimental Tests of Quantum Effects in Cytoskeletal Proteins," Chapter 4 of The Emerging Physics of Consciousness, ed. Jack A. Tuszynski. The Frontiers Collection. N. Y.: Springer, Berlin, Heidelberg, 2006. 95-170. (Invited book chapter.)
A. M. Woodward, A. Jones, X.-Z. Zhang, J. J. Rowland, and D. B. Kell. "Rapid and Non-invasive Quantification of Metabolic Substrates in Biological Cell Suspensions Using Nonlinear Dielectric Spectroscopy with Multivariate Calibration and Artificial Neural Networks. Principles and applications," Bioelectrochem. Bioenerg. 40 (1996): 99-132. 19 W. Khlbrandt. "Structure, and Mechanism of P-type ATPases," Nat. Rev. Mol. Cell Biol. 5 (2004): 282-95. 20 A. M. Woodward and D. B. Kell. "Confirmation Using Mutant Strains that the Membrane-bound H+-ATPase is the Major Source of Nonlinear Dielectricity in Saccharomyces cerevisiae," FEMS Microbiol Lett. 84 (1991): 91-96. 21 D. Nawarathna, J.R. Claycomb, J.H. Miller, Jr. and M.J. Benedik. "Nonlinear Dielectric Spectroscopy of Live Cells Using Superconducting Quantum Interference Devices," Appl. Phys. Lett. 86 (2005): 0239021-3. 22 K. Asami, "Characterization of Biological Cells by Dielectric Spectroscopy," J. of Non-crystaline Solids 305 (2002): 268-77.
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Presentations Nawarathna, D., J. Gardner, G. Cardenas, J. R. Claycomb, J. H. Miller, Jr., and W. R. Widger, "Electromagnetic Probing of Mitochondria and Chloroplasts Reveals Unique Harmonics Due to Specific Components of the Electron Transport Chain," Biophysical Society 50th Annual Meeting, Feb. 1820, 2006, Salt Lake City, UT. Nawarathna, Dharmakirthi, Jeffrey Gardner, Gustavo Cardenas, David Warmflash, John Miller, William Widger, and James Claycomb, "Nonlinear Electromagnetic Responses of Active Molecular Motors in Live Cells and Organelles," Bull. Am. Phys. Soc. 51, 200 (2006), March Meeting of the American Physical Society, Session B29, Focus Session on Microorganism Motility, March 1317, 2006; Baltimore, MD. Vajrala, Vijayanand, James Claycomb, and John H. Miller, Jr., "Analytical Model of Induced Transmembrane Potentials in Cells and Organelles," Bull. Am. Phys. Soc. 51, 1524 (2006), March Meeting of the American Physical Society, Session Y26, Focus Session on the Physics of Physiological Systems, March 1317, 2006; Baltimore, MD.
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Miller, J. H., Jr., D. Warmflash, D. Nawarathna, J. Gardner, G. Cardenas, and W. R. Widger, "Low-Frequency Electromagnetic Probes of Live Organisms," Session on Biogeophysics, 2006 Joint Assembly between the American Geophysical Union, Geochemical Society, Microbeam Analysis Society, Mineralogical Society of America, Society of Exploration Geophysicists, and Unin Geofsica Mexicana, May 2326, 2006, Baltimore, MD (Invited talk). Funding and Proposals Miller, John H., Jr. "Dielectric Spectroscopy of Chemical and Biological Systems," Robert A. Welch Foundation, June 1, 2004May 31, 2007. $165,000. Miller, John H. Miller, Jr., PI, William R. Widger, Co-I, "Noninvasive Sensors of Metabolic Activity," NIH, RFA-HL07-007, Bioengineering Approaches to Energy Balance and Obesity (R21), $150K/yr of direct costs requested for 3 years. Additional co-investigators/collaborators include Dale J. Hamilton, MD, and Richard J. Robbins, MD, of Methodist Hospital. Miller, John H., Jr., PI, "Nonlinear Impedance Spectroscopy of Chemical and Biological Systems," Robert A. Welch Foundation, renewal of E-1221, $60,000/year direct costs requested for three years. Funding Initiative NIH (R01), United Mitochondrial Disease Foundation and American Diabetes Assoc.: Marin Laughlin, an NIDDK program director in NIH, and others in NIBIB and NHLBI have expressed considerable interest in UH ideas and methodology for detecting mitochondrial function.
SPECTROSCOPY--Dr. John H. Miller Jr. supervises Shih-Ying Hsu, a Taiwanese doctoral student in biophysics, in a study of electromagnetic responses of living cell suspensions.
SCIENCE AND ENGINEERING--The Science and Engineering Research and Classroom Complex on the UH main campus is a 200,000-sq. ft. facility offering five floors of laboratory space capable of supporting an estimated 40 research laboratories.
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