University of Houston • University of Houston-Clear Lake • ISSO Annual Report Y2004 • 67-72
Martian Soil Biosensors Based on Dielectric Spectroscopy
Abstract--UH researchers explore new biosensing techniques, including dielectric spectroscopy and nonlinear harmonic response, which could ultimately be employed to develop instruments to test for the presence of living organisms in samples from outer terrestrial bodies. Our results suggest that dielectric spectroscopy at variable temperatures can distinguish live organisms from non-living complex macromolecules and may eventually be suitable for in situ astrobiology studies on the surface of Mars or, eventually, in the liquid ocean beneath the ice of Europa. Finally, we have recently discovered resonant-like behavior in the frequency-dependent harmonic responses of live cells. Preliminary evidence suggests that this behavior may result from active molecular motor complexes unique to live organisms.
The question of whether or not life once existed on Mars, or perhaps still exists today, has been of considerable interest since the nineteenth century. This issue has profound scientific implications for the evolution of life on Earth and the distribution of life in the cosmos.1 The Viking program, in 1976, made the first serious attempt to detect the presence of living or fossilized organisms in Martian soil and yielded ambiguous, somewhat negative results.2 However, recent studies of the Martian meteorite Allan Hills 84001 (ALH84001) suggest that microbial life existed on Mars about four billion years ago.3 Perhaps the most compelling evidence is the presence of magnetite (Fe3O4) crystals found within 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 known or 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 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 make these exciting candidates for the
existence of extraterrestrial life in our solar system.
Current detection and characterization systems for the presence of life forms are mainly
based either on: (1) gas chromatography, mass spectroscopy, and immunoassays for the
identification of organic materials and key signature biological macromolecules or (2)
cell culture, flow cytometry, polymerase chain reaction (PCR), sequencing, and nucleic
acid targeted hybridization for the identification of organisms at the genus and species
level. However, these techniques are expensive and time-consuming, and generally require
extensive knowledge and skills in molecular biology. Therefore there is a need to develop
a small portable, low cost, low power, an easy-to-use device, which could be taken on
robotic missions and operated by astronauts. The technique under investigation here, based
on dielectric properties of regolith soils and extracts, could ultimately lead to the
development of a small, lightweight, low power instrument package suitable for robotic
mission opportunities in the 2011 or 2013 timeframe, and for human missions beyond that
time.
Goal of the Project: The goal of this project is to investigate dielectric spectroscopy12,13 and related methods (e.g., nonlinear harmonic response14) as possible techniques for the detection of live organisms. One objective is to characterize Martian soil simulants using dielectric spectroscopy. A challenge for astrobiological investigation of Mars and other extraterrestrial bodies is to develop in situ instruments capable of distinguishing environmental samples or extracts containing life forms from those that do not. The life-detection technology must not be geocentric; that is it must not be targeted to characteristics that, although specific to life, may be limited to those life forms native to Earth. We are thus investigating dielectric spectroscopy (DS) as a life detection tool because life throughout the Cosmos, regardless of its biochemistry and the nature of its genetic material, must utilize a variety of complex, charged macromolecules.
Methodology
The dielectric constant e(w) of
a material is a measure of its linear response to an applied ac electric field at a
frequency w. This property is determined by the motion of
free charges inside the live cells, as well as the way macromolecules polarize in response
to the applied field. In our experiments, we employ a parallel plate capacitor
configuration for linear response measurements, as described in the next section, and a
four-probe method to measure any nonlinear harmonics produced by changes in the
conformational states of macromolecular enzyme complexes.
Three distinct dispersions (or relaxations), termed a-, b-, and g-dispersions, can be measure in the linear dielectric responses of biological cell suspensions and tissues over the frequency range 1 Hz-10 GHz. The a-dispersion, which appears below several kHz, is unique to living organisms and has been found to correlate with the cellular membrane potential.15,16 The b-dispersion, typically observed at MHz frequencies, is due to interfacial polarization and is mainly attributed to the insulating plasma membrane surrounding each cell. The g-dispersion, which lies above 1 GHz,17 results from reorientation of water and biological macromolecules. Our experiments on linear dielectric response primarily focus on the a- and b-responses, and employ a Solartron Impedance Analyzer, which measures complex admittance at frequencies up to 32 MHz. In addition, we employ related methods, such as nonlinear harmonic response, to probe signals produced by active molecular motors that are unique to live organisms, using a Stanford Research SR 780 Vector Signal Analyzer.
Equipment and Special Technology
A diagram of our setup for linear dielectric response, using a liquid capacitor cell and a
Solartron Analytical Model 1260 Impedance Analyzer, is shown in Fig. 1. This setup enables
us to obtain the complex dielectric response, with both the real and imaginary parts,
where the imaginary part of the dielectric response is proportional to the conductivity
divided by the frequency.
Figure 1. Setup used to measure linear dielectric response (top view, upper left; side view, at right). The liquid capacitor cell consists of two stainless steel electrodes (20-mm diameter) with a precision digital micrometer controlling the plate separation (to 3-mm precision), a teflon liquid sample holder (for up to 1 ml samples), and a guard ring to reduce fringe effects. The two electrodes are connected to the impedance analyzer (Solartron Analytical Model 1260) via the dielectric interface. The medium between the two electrodes can be modeled as a capacitance in parallel with a resistance, as shown at the lower left.
Results and Discussion
In an initial study, as environmental samples, we used common soil and JSC Mars-1, a
volcanic ash from Hawaii, developed for use as a Mars regolith simulant.18 Biologically
active, JSC Mars-1 contains microorganisms and biomolecules equivalent to 106-107
cells/gram,19 less than common soils (which can contain quantities of up to 109
cells/gram). Portions of each environmental sample were left untreated, while other
portions were sterilized: autoclaved for 60 minutes at 121°C, at 2 atm, then heated in an
oven at 220°C for 3 hours followed by exposure to ultraviolet light for 16 hours. Water
extractions were then performed on sterilized and untreated soil and JSC Mars-1 samples.
Extracts of untreated soil and JSC Mars-1 yielded multiple microbial strains when
incubated on Luria-Bertani (LB) agar for 24 hours at three temperatures: 23°C, 30°C, and
37°C (see Fig. 2). Extracts of sterilized soil and JSC Mars-1 showed no growth at any of
these temperatures, indicating that the sterilization protocol had indeed destroyed all
living forms within the environmental samples.
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Figure 2. (Top) LB agar plates incubated with
extract from un-sterilized soil and JSC Mars-1 regolith simulant.
(Bottom) LB agar plates incubated with extract from sterilized soil and JSC
Mars-1 regolith simulant.
When DS was conducted on extracts, the dielectric constant and conductivity were found to be higher for sterilized samples as compared with untreated samples (Fig. 3). We hypothesize that the sterilization protocol results in increased dielectric constant and conductivity due to lysis of cells and consequent release of charged molecules. However, the values obtained for unsterilized samples may be due not only to the presence of charged molecules, but also to membrane potentials of living cells. Samples containing living cells may thus be distinguishable from those containing only macromolecules by performing DS at variable temperatures.
Figure 3. Relative dielectric constants (top, real part) and conductivities (bottom) of several samples, as indicated.
Thus at two temperatures, 4°C and 37°C, we tested a suspension of the bacterium, E. coli (2.5 × 109 cells/ml, see Fig. 4), as well as three examples of large, charged biomolecules: deoxyribonucleic acid (DNA), hemoglobin (Hb), and bovine serum albumin (BSA). At 10 Hz, dielectric constant for E. coli suspension increased by 70% at 37°C as compared to 4°C, while dielectric constants for DNA, Hb, and BSA increased by 28%, 17%, and 49% respectively. However, the DNA, Hb, and BSA used in this preliminary study were then found to be contaminated with microorganisms, and as in the case of E. coli, the effects of temperature on life may be the reason the dielectric differences measured for these compounds. We therefore used fetal bovine serum (FBS) (containing proteins, cell wall lipids, and other compounds), specially treated to eliminate all known or suspected life forms, including the controversial entities known as nanobacteria.20,21 The results show that at 10 Hz the dielectric constant for sterile FBS increases by only approximately 6.5% for the FBS at 37°C vs. 4°C, as shown in Fig. 5.
Figure 4. Relative dielectric constants (top, real part) and conductivities (bottom) of a suspension of E. coli (2.5 × 109 cells/ml) at 4°C and 37°C.
Figure 5. Relative dielectric constants (top, real part) and conductivities (bottom) of a specially sterilized sample of Fetal Bovine Serum (FBS) at 4°C and 37°C. Note that little change with temperature is observed.
Conclusions
Our results indicate that varying the temperature increases the ability to differentiate
live organisms from non-living complex biomolecules using dielectric spectroscopy (DS). DS
at variable temperatures may thus be applicable to in situ astrobiology studies
on the surface of Mars or, eventually, in the liquid ocean beneath the ice of Europa.
Additionally, other manipulations, such as treating samples with stereoisomers of simple
nutrients (i.e., small amino acids or sugars) prior to dielectric spectroscopy, may also
help in distinguishing biological from sterile samples. More recently, we have discovered
resonant-like peaks in the frequency-dependent nonlinear harmonic responses of live cells,
and preliminary evidence suggests that this behavior may result from active molecular
motor complexes unique to live organisms, thus providing another tool for detecting life
forms and for fundamental research in biophysics.
References
1B. M. Jakosky and E. L. Shock, "The Biological Potential of Mars, the
Early Earth, and Europa," J. Geophys. Res. 103 (1998): 19,359-64.
2L. 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.
3D. 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.
4K. 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 Hexa-Octahedral Magnetite Crystals in ALH84001: Presumptive
Biosignatures," Proc., Nat. Acad. Sci. USA 98 (2001): 2164-69.
5K. 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 Biosignature in the Martian Meteorite
ALH84001," Applied & Environmental Microbiology 68 (2002): 3663-72.
6B. P. Weiss, Y. L. Yung, and K. H. Nealson, "Atmospheric Energy for
Subsurface Life on Mars?" Proc., Nat. Acad. Sci. USA 97 (2000): 1395-99.
7M. 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.
8M. H. Carr, Water on Mars. New York: Oxford Univ. Press, 1996.
9W. B. Whitman, D. C. Coleman, and W. J. Wiebe, "Prokaryotes: The Unseen
Majority," Proc., Nat. Acad. Sci. USA 95 (1998): 6578-83.
10K. H. Nealson, "The Limits of Life on Earth and Searching for Life on
Mars," J. Geophys. Res. 102 (1997): 23,675-86.
11J. 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.
12H. P. Schwan, "Electrical properties of tissue and cell
suspensions," in Advances in Biological and Medical Physics. Vol 5. Eds. J.
H. Lawrence and C. A. Tobias. New York: Academic Press, 1957. 147-209.
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.
15C. Prodan, "Dielectric Properties of Live Cell Suspensions," Ph.D.
Dissertation, University of Houston, 2003.
16C. Prodan, F. Mayo, J. R. Claycomb, J. H. Miller, Jr., and M. J. Benedik,
"Low-Frequency, Low-Field Dielectric Spectroscopy of Living Cell Suspensions,"
J. Appl. Phys. 95 (2004): 3754-56.
17J. B. Hasted, Aqueous Dielectrics. London: Chapman and Hall, 1973.
18C. C. Allen, R. V. Morris, K. M Jager, D. C. Golden, D. J. Lindstrom, M. M.
Lindstrom, and J. P. Lockwood, "Martian Regolith Simulant JSC Mars-1," 29th
Annual Lunar and Planetary Science Conference, NASA Johnson Space Center, Houston, TX,
March 16-20, 1998.
19C. C. Allen, C. Griffin, A. Steele, N. Wainwright, and E. Stansbery,
"Microbial Life in Martian Regolith Simulant JSC Mars-1," 31st Lunar and
Planetary Science Conference, Johnson Space Center, Houston, TX, March 13-17, 2000.
20E. O. Kajander, N. Ciftcioglu, K. Aho, and E. Garcia-Cuerpo,
"Characteristics of Nanobacteria and Their Possible Role in Stone Formation," Urol.
Res. 2 (2003): 47-54.
21A. P. Sommer, H. I. Hassinen, and E. O. Kajander, "Light-Induced
Replication of Nanobacteria: A Preliminary Report," J. Clin. Laser Med. Surg.
5 (2002): 241-44.
Publications
Claycomb, J. R., D. Nawarathna, V. Vajrala, and J. H. Miller, Jr. "Power Law Behavior
in Chemical Reactions," J. Chemical Physics 121.24 (2004): 12428-30.
Claycomb, J. R., V. Vajrala, D. Nawarathna, and J. H. Miller, Jr. "Impedance
Magnetocardiography: Experiments and modeling," J. Applied Physics 96.12
(2004): 7650-54.
Martirosyan, K. S., J. R. Claycomb, J. H. Miller, Jr., and D. Luss. "Generation of
the Transient Electrical and Spontaneous Magnetic Fields by Solid State Combustion," J.
Applied Physics 96.8 (2004): 4632-36.
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. Biological Physics (accepted for publication, in press, 2005).
Nawarathna, D., J. R. Claycomb, J. H. Miller, Jr., and M. J. Benedik. "Nonlinear
Dielectric Spectroscopy of Live Cells Using Superconducting Quantum Interference
Devices," Applied Physics Letters 86 (2004): 023902-1-3.
Prodan, C., F. Mayo, J. R. Claycomb, J. H. Miller, Jr., and M. J. Benedik.
"Low-Frequency, Low-Field Dielectric Spectroscopy of Living Cell Suspensions," J.
Applied Physics 95.7 (2004): 3754-56.
Presentations
Miller, J. H., Jr. "Biosensors Based On Dielectric Spectroscopy," Smart Medical
Technologies (SMT) Summit 2004, NASA-JSC, April 7-8, 2004 (invited talk).
--. "Electromagnetic Properties of Live Cells and Proteins," Department of
Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, Oct. 19,
2004 (invited talk).
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,"
5th International Conference on Biological Physics-ICBP 2004, Chalmers University of
Technology & Göteborg University, Göteborg, Sweden, Aug. 23-27, 2004.
Miller, J., G. Cardenas, A. Garcia-Perez, W. More, and A. Beckwith. "Quantum Pair
Creation of Soliton Domain Walls," Bulletin of the American Physical Society
49.1, Part 2 (2004): 915, March Meeting of the American Physical Society, Montréal,
Canada, March 22-26, 2004.
Nawarathna, D., J. Claycomb, and J. Miller, Jr. "Electrical Impedance Spectroscopy of
Biological Cells," Bulletin of the American Physical Society 49.1, Part 2
(2004): 451, March Meeting of the American Physical Society, Montréal, Canada, March
22-26, 2004.
Nawarathna, D., J. R. Claycomb, H. Sanabria, D. Warmflash, and J. H. Miller, Jr.
"Nonlinear Dielectric Spectroscopy of Eucaryotic Cells," Biophysical Society
49th Annual Meeting, Long Beach, CA, Feb. 12-16, 2005,
Sanabria, H., D. Nawarathna, A. Mershin, J. H. Miller, Jr., D. V. Nanopoulos, A.
Kolomenski, H. A. Schuessler, and R. F. Luduena. "Dielectric Properties of Live Cells
and Protein Suspensions," Biophysical Society 49th Annual Meeting, Long Beach,
California, Feb. 12-16, 2005.
Sanabria, H., D. Nawarathna, and J. H. Miller, Jr. "Dielectric Properties of
Biological Cells and Protein Suspensions," Ninth Annual Structural Biology Symposium,
Sealy Center for Structural Biology, University of Texas Medical Branch at Galveston,
Galveston, TX, April 30-May 1, 2004.
Vajrala, V., D. Nawarathna, J. Claycomb, and J. H. Miller,
Jr. "Nested Sphere Model for SQUID-based Impedance Magnetocardiography,"
Bulletin of the American Physical Society 49.1, Part 2 (2004): 1128, March Meeting of
the American Physical Society, Montréal, Canada, March 22-26, 2004.
Vajrala, V., D. Nawarathna, J. R. Claycomb, and J. H. Miller,
Jr. "High-Tc SQUID-Based Impedance
Magnetocardiography," Applied Superconductivity Conference: ASC-2004, Jacksonville,
FL, Oct. 3-8, 2004.
Warmflash, D., J. H. Miller, Jr., D. S. McKay, G. E. Fox, and D. Nawarathna.
"Dielectric Spectroscopy for In-Situ Detection of Microbial Life Forms on
Mars," Mars Astrobiology Science and Technology Workshop, Carnegie Institution of
Washington, Washington, DC., Sept. 8-10, 2004.
Funding and Proposals
Miller, John 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, $757,726 requested,
$250,000 funded to date.
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,"
submitted to NASA-ASTEP (Astrobiology Science and Technology for Exploring Planets)
Program, pending, $895,277 requested for three years ($793,197 for UH, $102,080 for
NASA-JSC).
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