University of Houston • University of Houston-Clear Lake •
ISSO Annual Report Y2005 • 33-38
Martian Soil Biosensors Based on Dielectric Spectroscopy
John H. Miller, Jr., Jaroslaw Wosik, David S. McKay, Jeffrey A. Jones,
Fathi Karouia, David Warmflash, Dharmakeerthi Nawarathna
Abstract--The goal is to investigate novel biosensing techniques,
including dielectric spectroscopy and nonlinear harmonic response, which could ultimately
be employed to develop instruments capable of detecting live organisms in samples from the
outer terrestrial bodies of the solar system. Our previous results suggested that variable
temperature dielectric spectroscopy can distinguish live organisms from nonliving complex
macromolecules and may be suitable for in situ astrobiology studies on the
surface of Mars or, eventually, in the liquid ocean beneath the ice of Europa. More
recently, we have measured the frequency- and amplitude-dependent nonlinear harmonic
responses of live cells, mitochondria, and chloroplasts, coupled with activator and
inhibitor studies. Results provide compelling evidence that physiologically relevant
processes in active macromolecular enzyme complexes are responsible for observed induced
harmonics, thus providing additional signatures of live organisms.
The possible existence of life on Mars in the distant past
or at present1 has been of interest for well over a century. This issue has
important scientific implications for the evolution of life on earth and the distribution
of life in the cosmos. The Viking program, in 1976, made the first attempt to detect
evidence of living or fossilized organisms in Martian soil and yielded ambiguous, somewhat
negative results.2 More recent studies3 of the Martian meteorite
Allan Hills 84001 (ALH84001) suggest that microbial life existed on Mars about four
billion years ago. Compelling evidence includes the presence of magnetite crystals (Fe3O4)
found in carbonate globules and their associated rims in the meteorite.4 About
one fourth of these tens-of-nanometer sized magnetites are nearly identical to those
produced by magnetotactic bacteria on Earth and are not expected to be produced by abiotic
means. It has, therefore, been argued that these Martian magnetite crystals are in fact
magnetofossils, which, if true, would constitute evidence of the oldest life forms known.5
Additional findings suggest that subsurface Martian life could potentially survive even
today.6 There is abundant geological evidence that ice was once deposited in
the regolith, where it should still be present above mid-latitudes.7 This ice,
which probably extends several kilometers below the surface, could be a source of liquid
water near magmatic intrusions.8 On Earth, the biomass of subterranean
organisms may equal or exceed that at the surface.9 These organisms can live in
highly saline conditions at temperatures from 115°C to -20°C.10,11 Such
conditions might prevail beneath the surface in an aquifer or in a hydrothermal system.
For these and other reasons, there is considerable interest in developing new techniques
of detecting subsurface life on Mars. Moreover, the likelihood that oceans of liquid water
exist below the icy surfaces of Europa and other moons makes these exciting candidates for
the existence of extraterrestrial life in our solar system.
Goal of the Project
The goal of this project is to investigate dielectric spectroscopy12,13 and
related methods, especially nonlinear harmonic response,14 for the detection of
live organisms. Toward this end, the project aims to elucidate possible signatures of
active macromolecular complexes unique to living biological systems. Potential signatures
include unusual behavior, distinct from those of inanimate materials, in the frequency-
and temperature-dependent dielectric response, and in the induced nonlinear harmonic
response as a function of frequency and amplitude.
Methodology
The previous report discussed the results of 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 focus on a study of potential signatures in
the nonlinear harmonic response, which include signals produced by active molecular motors
unique to live organisms.
For measurements at kilohertz frequencies, we employ a four-electrode system, utilizing
a Stanford Research SR780 signal analyzer that we operate as a spectrum analyzer. 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 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 lay in allowing one to deconvolve the
effects of nonlinearities within the electrochemical system from those due to the
biological cells themselves.
Equipment and Special Technology
A diagram of our four-electrode setup for nonlinear response, including the Stanford
Research SR780, is shown in Fig. 1. This setup enables us to obtain the frequency- and
amplitude-dependence of harmonics induced by the sample (e.g., cell suspension) under
investigation. At low frequencies, we employ a superconducting quantum interference device
(SQUID) to directly probe the magnetic fields produced by the conduction and displacement
currents. This reduces spurious harmonics generated at the electrode-medium interface,
which become especially problematic at frequencies below 100 Hz. Details of this work have
been reported in our 2004 study in Applied Physics Letters.14
Results and Discussion
We measured the nonlinear harmonic response of suspensions of
budding yeast cells (S. cerevisiae), mitochondria (extracted from bovine heart
and mouse liver cells), B. indicas (a prokaryotic relative of the mitochondrial
ancestor), and chloroplasts (extracted from spinach) using the four-electrode setup shown
in Fig. 1, in which a sinusoidal voltage is applied across the two outer electrodes, while
the spectrum of induced harmonics is recorded across the two inner electrodes using a
spectrum analyzer. When a suspension of cells or extracted organelles is excited using a
single-frequency sinusoidal voltage excitation, a series of harmonics is produced for
sufficient ac field amplitudes. Any background harmonics due to the electrode interface
are subtracted out by measuring a reference medium with conductivity matched to that of
the cell suspension.
Figure 1 (r.). Four-electrode setup used to measure nonlinear
harmonic response. (a) Experimental setup with external function generator and SR780
signal analyzer. (b) Planar, top view of the four-electrode sample cell showing the
dimensions and geometry. The electrodes are 12-carat gold pins, 7 mm in length
(cross-sections shown in figure).
When measuring the magnitudes of the induced second and third harmonic amplitudes vs.
applied frequency for whole budding yeast cells, we observed two peaks that appeared to
grow out of the background as cell concentration is increased. In addition, we found that
rotenone, an inhibitor that affects complex I of the mitochondrial respiratory chain,
reduces the amplitudes of the harmonics. We observed even more dramatic suppressing
effects when adding potassium cyanide, a respiratory inhibitor that binds to the
cytochrome c oxidase complex and essentially shuts down the entire electron transport
chain on the mitochondrial inner membrane. Similar behavior is observed for B. indicas,
a relative of the mitochondrial ancestor. Respiratory inhibitors suppress the
mitochondrial transmembrane potential, hence shutting off the ATP-synthase molecular
turbine, and prevents the production of ATP. Importantly, however, yeast cells are not
killed by cyanide and remain capable of fermentation. In order to further study whether
bioenergetic processes play a major role, we have performed similar measurements on
isolated mitochondria extracted from mammalian cells.
Figure 2 shows the second harmonic response vs. applied frequency for uncoupled
mammalian mitochondria, in which complex II has been activated through the addition of
glutamate malate, whereas the ATP synthase complex is inactive due to the lack of a
transmembrane potential and proton gradient. These results suggest that the peak in Fig. 2
may be attributed to electron/proton transport processes occurring between complex II and
complex IV of the electron transport machinery. Note that the inhibitor rotenone quenches
the harmonic response, as shown in Fig. 2. We also observe a higher frequency peak,
appearing at applied frequencies of around 12 kHz in budding yeast cells and B.
indicas, a relative of the mitochondrial ancester. In both of these cases, the ATP
synthase complex is active, suggesting that the high frequency peak in these organisms may
result from coupling to the remarkable molecular turbine in the F0 unit of ATP synthase.
Figure 2. Second harmonic response vs. applied fundamental frequency of
uncoupled mitochondria, in which the electron transport chain is active (closed circles,
before adding the inhibitor rotenone) and inactive (open circles, after adding rotenone),
for an applied field amplitude of 5 V/cm. In uncoupled mitochondria, the ATP synthase
molecular turbine is inactive and a second, higher frequency (~12 kHz) peak, seen in
budding yeast and B. indicas, is absent in this case.
Photosynthetic organisms, including cyanobacteria and chloroplasts (responsible for
photosynthesis in plants), are potentially exciting organisms to study because they
utilize light rather than chemical energy to establish electrochemical gradients, allowing
for greater flexibility and precision in controlling experimental conditions. Figure 3
cites results obtained for chloroplasts, showing dramatic changes in harmonic response
with the presence or absence of light. These results strongly indicate that the observed
behavior is caused by, in this case, active processes due to photosynthesis, and provide
increased confidence of the physiological relevance of harmonic response measurements.
Note, however, that the frequency-dependence is rather different from that observed for
yeast cells and mitochondria, and may incorporate responses from the light harvesting
complexes, reaction centers, and photosystems.
Figure 3. Second harmonic response vs. applied fundamental frequency
for chloroplasts, with light and without light, for an applied field amplitude of
5 V/cm.
Conclusions
Our latest results suggest that ac electric fields, which capacitively couple through the
plasma membrane, can interrogate bioenergetic and other processes in internal cellular
organelles and potentially identify signatures of life. Additional experiments are planned
to study coupled mitochondria, which should enable further confirmation of any signals
arising from biological energy-transducing complexes. The observed unusual behavior, which
in the case of chloroplasts is activated by light, provides signatures unique to live
organisms and could ultimately be applied to in situ astrobiology studies on the
surface of Mars or, eventually, in the liquid ocean beneath the ice of Europa. Additional
terrestrial applications include fundamental research in biophysics, studies of the
effects of drugs on live cells to aid in pharmaceutical development, studies of cancer
cells, and the development of medical diagnostic instrumentation.
References
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11J. C. Priscu, C. H. Fritsen, E. E. Adams, S. J. Giovannoni, H. W. Paerl, C.
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12H. P. Schwan, "Electrical Properties of Tissue and Cell
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13K. Asami, "Characterization of Biological Cells by Dielectric
Spectroscopy," J. Non-Crystaline Solids 305 (2002): 268-77.
14D. 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 (2004): 023902-1-3.
Publications
Miller, J. H., Jr., D. Nawarathna, D. Warmflash, F. A. Pereira, and W. E. Brownell.
"Dielectric Properties of Yeast Cells Expressed with the Motor Protein Prestin,"
J. Biol. Phys. 31.3-4 (2005): 465-75.
Miller, J. H., Jr., D. Nawarathna, V. Vajrala, J. Gardner, and W. R. Widger.
"Electromagnetic Probes of Molecular Motors in the Electron Transport Chains of
Mitochondria and Chloroplasts," J. de Physique IV (France) 131 (2005):
363-66.
Nawarathna, D., J. H. Miller, Jr., J. R. Claycomb, G. Cardenas, and D. Warmflash.
"Harmonic Response of Cellular Membrane Pumps to Low Frequency Electric Fields,"
Phys. Rev. Lett. 95 (2005): 158103-1-4.
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. (Submitted.)
Sanabria, H., 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," Biophysical J. (Accepted for publication).
Vajrala, V., D. Nawarathna, J. R. Claycomb, and J. H. Miller, Jr. "Impedance
Magnetocardiography Using High-Tc SQUIDs," IEEE Trans. on Applied
Superconductivity 15.2 (2005): 680-83.
Presentations
Miller, J. H., Jr. "Condensed Matter Physics Phenomena in Biological Systems,"
Texas Center for Superconductivity, University of Houston, April 21, 2005. (Invited.)
---. "Electromagnetic Properties of Live Cells and Proteins," Los Alamos
National Laboratory, National High Magnetic Field Laboratory, Los Alamos, NM, July 20,
2005. (Invited.)
---. "Electromagnetic Sensors of Nanoscale Biological Motors," Associated
Nanotechnology Congress, Strategic Partnership for Research in Nanotechnology, 3rd Annual
SPRING Conf., Rice U., Houston, TX, Oct. 10-11, 2005. (Invited.)
---. "Resonant Peaks in the Nonlinear Electromagnetic Responses of Live Cells and
Mitochondria," Department of Bioengineering, Rice U., Houston, TX, Feb. 7, 2005. (Invited).
Miller, J. H., Jr., D. Nawarathna, H. Sanabria, V. Vajrala, and J. R. Claycomb.
"Nonlinear Harmonic Responses of Live Cells Using High-Tc Superconducting Quantum
Interference Devices," 24th Intl. Conf. on Low Temperature Physics, Orlando, FL, Aug.
10-17, 2005.
Miller, J. H., Jr., D. Nawarathna, V. Vajrala, J. Gardner, and W. R. Widger.
"Electromagnetic Probes of Molecular Motors in the Electron Transport Chains of
Mitochondria and Chloroplasts," Intl Workshop on Electronic Crystals, Cargèse,
France, Aug. 21-27, 2005. (Invited for expanded presentation.)
Nawarathna, D., D. Warmflash, J. H. Miller, Jr., and J. Claycomb. "Noninvastive
Probes of Mitochondrial Molecular Motors," Bull. Am. Phys. Soc. 50 (2005): 1342.
Meeting of the American Physical Society, Los Angeles, CA, March 21-25, 2005.
Nawarathna, D., J. Gardner, G. Cardenas, J. R. Claycomb, J. H. Miller, Jr., and W. R.
Widger. "Electromagnetic Probes of Molecular Motors in the Electron Transport Chains
of Mitochondria and Chloroplasts," Associated Nanotechnology Congress, Strategic
Partnership for Research in Nanotechnology, 3rd Annual SPRING Conf., Rice U., Houston, TX,
Oct. 10-11, 2005.
Vajrala, V., J. Claycomb, and J. Miller. "Modeling Studies of Induced Internal
Transmembrane Potentials and Molecular Motors in Live Cells," Bull. Am. Phys. Soc.
50 (2005): 1342-43. Meeting of the American Physical Society, Los Angeles, CA, March
21-25, 2005.
Funding and Proposals
Miller, J. H., Jr. "Dielectric Spectroscopy of Chemical and Biological Systems,"
Robert A. Welch Foundation, June 1, 2004-May 31, 2007, $165,000.
Miller, J. H., Jr. and J. Wosik. "Dielectric Spectroscopy of Biological Agents,"
DARPA/Naval Surface Warfare Center, Sept. 26, 2003-Sept. 25, 2005, $250,000.
Miller, J. H., Jr., D. S. McKay, G. E. Fox, J. Wosik, and D. Warmflash. "Biosensors
Based on Dielectric Response: A Non-Geocentric Approach for In Situ Life Detection,"
NASA-Astrobiology Science and Technology for Exploring Planets Program. Requested for
three years ($793,197 for UH, $102,080 for NASA-JSC), $895,277. (Pending.)
In 2006 we intend to submit at least one NIH (R01 or R21) proposal, likely in
biomedical engineering or a related area, which will build on our recent results and
publications on noninvasive electromagnetic biosensors.
