Detecting past life on Mars
SwRI researchers develop new technique to identify biomarkers
By Michael Miller, Darrell Dunn and Narasi Sridhar, Ph.D.
The possibility of life on Mars has fascinated mankind since the discovery of canal-like features on the planet by Giovanni Schiaparelli in 1877. He, and later Percival Lowell, propagated a theory that they were canals cut by beings of higher intelligence. Belief in Martian-made canals began to fade at the beginning of the 1900s, and they were debunked by NASA’s Mariner 6 and 7 missions in 1969.
Then, Mariner 9 in 1971 showed riverbed-like natural features on the Martian surface that suggested outflows of lava or water. The Viking missions, which launched in 1975 and reached Mars in 1976, were the first to land scientific instruments on the planet’s surface. The instrumentation included a biology instrument, gas chromatograph-mass spectrometer, X-ray fluorescence spectrometer and other soil property measuring instruments. The biology instrument was designed to extract organic molecules by heating Martian soil samples to 500 degrees Centigrade and analyzing the volatile species using the gas chromatograph-mass spectrometer. The results were inconclusive, perhaps because the technique lacked sensitivity. Since then, scientists have come to believe that the Martian atmosphere is self-sterilizing because of its extreme dryness and the oxidizing nature of the chemistry caused by ultraviolet radiation. Therefore, signs of past life, or biomarkers, may be in the form of degraded organic molecules.
The Mars Pathfinder mission landed the Sojourner rover on Mars in 1997. Sojourner contained an alpha proton X-ray spectrometer that analyzed rock and soil composition. In January 2004, two rovers, Spirit and Opportunity, landed on two different sides of Mars to conduct detailed visual and microscopic examinations of the soil and surface features. These rovers carried identical instrumentation packages including the mini-Thermal Emission Spectrometer (mini-TES), which provided information on molecular composition of rocks and soils, the Alpha-Particle X-ray Spectrometer (APXS), which yielded elemental compositions of the rocks, and a Mössbauer spectrometer, which provided information on the abundance of iron-containing minerals. All these instruments helped lead scientists to the conclusion that water existed at one time on the surface of Mars. However, no instrumentation packages thus far have provided any direct evidence of water or organic substances symptomatic of life, past or present.
Since the Viking Lander mission in 1976, assessments have been made of the detectability of organic molecules indicative of past or present life on Mars. More recently, a team headed by Scripps Oceanography Institute is developing an instrument package that relies on pulverizing Martian soil, extracting the powder in water, separating the dissolved organic species through a capillary electrophoresis technique, and analyzing them using a number of methods. Although extracting, volatilizing and analyzing organic samples holds promise in identifying past life on Mars, the technique has some limitations. Because of the presence of high ultraviolet radiation and the resulting hydrogen peroxide and other free radicals, the organic compounds arising from biological activity may have been long ago converted to various benzocarboxylic acids. These acids could have then combined with iron oxides present on Mars’ surface to form various nonvolatile carboxylate salts. Additionally, dilution of these salts caused by soil movements and wind may further elude detection of trace concentrations (on the order of a few hundred parts per billion) of surface organic species resulting from past biological activity. Finally, some organic molecules are not easily extracted. What is needed, therefore, is a complementary technique that can directly identify organic molecules embedded in soils and rocks.
Raman spectroscopy has been proposed to identify not only the mineralogy on Mars, but also organic molecules present within the soil and rocks. Raman spectroscopy relies on impinging a laser beam of known wavelength on a sample and analyzing the shift in wavelength of the scattered laser light. The vibrations of the molecules on the sample subtly affect the wavelengths of the scattered laser light, which, if analyzed, can yield information about the type of molecule present.
However, a major limitation of Raman spectroscopy is its weak signal strength. The intensity of the Raman scattered wavelength is less than a millionth of the intensity of a Rayleigh scattered laser light (that is, laser light scattered without a shift in wavelength). For this reason, extremely sensitive detection techniques are required even for molecules that are relatively abundant. To detect organic molecules originating from biomarkers present in extremely small concentrations and mixed in with minerals in soils, conventional Raman spectroscopy cannot be used.
Surface-Enhanced Raman Spectroscopy
Martin Fleischmann and his colleagues in 1974 stumbled on an observation that organic molecules showed an almost million-fold increase in Raman scattering intensity when present on a roughened silver surface. This discovery has since led to the use of surface-enhanced Raman spectroscopy (SERS) as a routine analytical tool for studying very small concentrations of molecules, down to single-molecule levels, on surfaces.
Under contract to NASA, researchers at Southwest Research Institute (SwRI) were tasked with identifying different SERS techniques suitable for biomarker analogues under typical Mars conditions of low temperatures and pressures. Tests conducted at SwRI showed that SERS is capable of observing femtomole level (10-15 moles) of an organic compound, benzotriazole, on a quartz mineral. With refinement of this technique, less than a femtomole of organic substances on minerals can be detected using SERS. Without a SERS-active surface, such a small amount cannot be observed by Raman spectroscopy. Because of this, the SwRI team focused on SERS as the technique capable of identifying the presence of biomarkers on Mars.
SERS has been used not only with silver, but also gold, copper, lithium and many other metals. Obtaining such an enormous intensification of Raman signal requires intimate contact between the metal particle and the molecule of interest, appropriate size and shape of the metal or other SERS-producing particles, and matching of the incident wavelength with the electronic properties of the particle. A well-established method of obtaining SERS is to have a ready-made surface of silver or gold nanoparticles and then deposit the sample to be analyzed on this surface. In such an arrangement, the sample to be analyzed has to be processed so that the molecule of interest can be extracted using a solvent. The target molecule dissolved in the solvent is deposited on the SERS surface, and the solvent is evaporated. However, not all molecules can be easily extracted in a solvent, and it may be useful to determine the association of a certain biomarker with a mineral to ascertain its origins.
The SwRI team developed a technique called inverted SERS, or iSERS. In iSERS, silver or gold nanoparticles are deposited directly onto the mineral sample to be analyzed instead of extracting the compound of interest in a solvent that is deposited on a prepared SERS substrate.
The team members also found that using iSERS allowed them to study the degradation of biomarkers on mineral surfaces under ultraviolet radiation. For example, both benzotriazole (BTA), a test molecule known to be SERS-active, and scytonemin, a pigment that is present in plants and may be relevant to the study of previous life on Mars, resist UV degradation on the mineral olivine, whereas benzoic acid degrades rapidly. Thus iSERS measurements can help researchers understand the fate of biomarkers under Mars-like conditions.
In developing this technique, it was critical that the process be tailored to the environmental conditions, including the pressure and range of temperatures expected on Mars. The SwRI team constructed a vacuum chamber to simulate the Martian atmosphere and Mars-like pressures to determine whether iSERS can be carried out by a rover operating on the Martian surface. Using the vacuum chamber, researchers simulated the Martian atmospheric conditions (approximately 10 millibars with essentially 95 percent CO2), injected silver nanoparticles in the form of a colloidal solution onto the mineral substrate positioned in the chamber, and then performed Raman spectroscopy in situ.
Of course, the type of life that may have existed on Mars is highly speculative. Simple plant species, such as algae, would be the most likely and would probably need to exist to support more complex life forms. Many organic compounds may be present on Mars, but only organic species that are UV-stable would be present under the current conditions. The SwRI team’s results suggest that Raman spectroscopy may be a useful analytical method for future rovers to detect trace concentrations of organic species and potential biomarkers, such as organic pigments, on minerals expected to be present on Mars.
The iSERS technique developed for NASA has already yielded other benefits. For example, for the U.S. Marine Corps Corrosion Prevention and Control (CPAC) program, SwRI researchers have used this technique to determine how volatile corrosion inhibitors (VCI) function. VCIs are used to protect electronics in enclosures such as lamp assemblies, and their in-situ performance under real-life exposure conditions has never been monitored. The iSERS technique can also be used to examine fracture surfaces to obtain a better understanding of the mechanism of cracking by detecting trace concentrations of species that may promote specific environmental-assisted cracking phenomena. At present, techniques such as scanning electron microscopy linked with energy-dispersive spectroscopy yield information only on elemental compositions. Understanding which molecular species are associated with a fracture can assist in the determination of appropriate solutions to problems.
Published in the Summer 2007 issue of Technology Today®, published by Southwest Research Institute. For more information, contact Joe Fohn.