Life Marker Chip Research Articles

Searching for life on Mars: degradation of surfactant solutions used in organic extraction experiments.

Life-detection instruments on future Mars missions may use surfactant solutions to extract organic matter from samples of martian rocks. The thermal and radiation environments of space and Mars are capable of degrading these solutions, thereby reducing their ability to dissolve organic species. Successful extraction and detection of biosignatures on Mars requires an understanding of how degradation in extraterrestrial environments can affect surfactant performance. We exposed solutions of the surfactants polysorbate 80 (PS80), Zonyl FS-300, and poly[dimethylsiloxane-co-[3-(2-(2-hydroxyethoxy)ethoxy)propyl]methylsiloxane] (PDMSHEPMS) to elevated radiation and heat levels, combined with prolonged storage. Degradation was investigated by measuring changes in pH and electrical conductivity and by using the degraded solutions to extract a suite of organic compounds spiked onto grains of the martian soil simulant JSC Mars-1. Results indicate that the proton fluences expected during a mission to Mars do not cause significant degradation of surfactant compounds. Solutions of PS80 or PDMSHEPMS stored at -20 °C are able to extract the spiked standards with acceptable recovery efficiencies. Extraction efficiencies for spiked standards decrease progressively with increasing temperature, and prolonged storage at 60°C renders the surfactant solutions ineffective. Neither the presence of ascorbic acid nor the choice of solvent unequivocally alters the efficiency of extraction of the spiked standards. Since degradation of polysorbates has the potential to produce organic compounds that could be mistaken for indigenous martian organic matter, the polysiloxane PDMSHEPMS may be a superior choice of surfactant for the exploration of Mars.

Development of extraction protocols for life detection biosensor-based instruments

Extraction experiments were performed to evaluate suitable sample-extraction and processing protocols that will be used by bioanalytical instruments like Life Marker Chip (LMC). These instruments will focus on the detection of molecules associated with life that will be extracted from the Martian soil. LMC is an antibody microarray biosensor instrument with optical readout, which uses fluorescently labeled antibodies, to detect and quantify the presence of polar and non-polar biomolecules, extracted from the Martian soil. The success of the LMC biomarkers detection experiment is dependent on the sample extraction protocol. The currently recommended extraction procedure, optimized for aliphatic biomarkers, consists in sonicating the samples using as solvent system 20:80 methanol:water with 1.5mg mL−1 tween 80, which respects all the tight requirements associated with LMC biosensor. Examining this procedure for a particular class of biomarkers, the nucleobases adenine, cytosine, uracil and hypoxanthine adsorbed onto magnesium oxide (MgO), we observed low extraction efficiency, confirming that the selected solvent system is not able to extract with high efficiency aromatic biomarkers.

Searching for biomolecules on Mars: Considerations for operation of a life marker chip instrument

The search for life on Mars requires instruments that detect organic matter and discriminate between potential sources. One such instrument is the life marker chip that recognizes small molecules which are characteristic of particular organic provenances. The use of an antibody-based detection system requires the delivery of small organic compounds in a suitable solvent. Dedicated extraction protocols have been developed partly through the use of a life marker chip breadboard system. Techniques which provide the strong diagnostic potential of the life marker chip necessitate appropriate sample types. Clay mineral-rich rocks are attractive targets owing to their (i) association with liquid water, (ii) propensity for organic matter and clay mineral co-deposition following transport from a wide hinterland, and (iii) relatively large surface area and therefore potential for trapping/adsorption of organic materials. The most appropriate target organic compounds are the hydrocarbon-dominated lipids that can be highly diagnostic and have relatively high preservation potentials. The sample sites on Mars and sample preparation steps that are needed for successful detection require careful consideration. In this paper we explore the scientific results that may be obtained through the operation of a life marker chip instrument on Mars.

Survivability of Immunoassay Reagents Exposed to the Space Radiation Environment on board the ESA BIOPAN-6 Platform as a Prelude to Performing Immunoassays on Mars

The Life Marker Chip (LMC) instrument is an immunoassay-based sensor that will attempt to detect signatures of life in the subsurface of Mars. The molecular reagents at the core of the LMC have no heritage of interplanetary mission use; therefore, the design of such an instrument must take into account a number of risk factors, including the radiation environment that will be encountered during a mission to Mars. To study the effects of space radiation on immunoassay reagents, primarily antibodies, a space study was performed on the European Space Agency's 2007 BIOPAN-6 low-Earth orbit (LEO) space exposure platform to complement a set of ground-based radiation studies. Two antibodies were used in the study, which were lyophilized and packaged in the intended LMC format and loaded into a custom-made sample holder unit that was mounted on the BIOPAN-6 platform. The BIOPAN mission went into LEO for 12 days, after which all samples were recovered and the antibody binding performance was measured via enzyme-linked immunosorbent assays (ELISA). The factors expected to affect antibody performance were the physical conditions of a space mission and the exposure to space conditions, primarily the radiation environment in LEO. Both antibodies survived inactivation by these factors, as concluded from the comparison between the flight samples and a number of shipping and storage controls. This work, in combination with the ground-based radiation tests on representative LMC antibodies, has helped to reduce the risk of using antibodies in a planetary exploration mission context.

Potential failure of life detection experiments on Mars resulting from adsorption of organic compounds on to common instrument materials

Some life detection instruments under development for operation on Mars use solvents to extract organic compounds from samples of Martian regolith and rock and to transfer the extracts to dedicated detectors. However, it is possible that organic compounds extracted from Martian samples and dissolved in the solvent could adsorb to instrument surfaces, potentially resulting in a failure to detect organic matter that could have been avoided by using more appropriate instrument materials. If successful detection and characterisation is to take place it is therefore essential to understand the interactions between dissolved organic targets and the surfaces of space instrument components. One such life detection instrument is the Life Marker Chip (LMC) being developed for the ExoMars mission, which relies on a novel surfactant-based solvent system and antibody-based detectors. We have tested the ability of a range of materials, including titanium, stainless steel, aluminium, the fluoropolymer Viton™, polytetrafluoroethylene (PTFE), nylon, polypropylene, polyethersulfone and cellulose acetate to adsorb a range of organic standards from the surfactant solution intended to be used by the LMC. Results indicate that aromatic hydrocarbons, specifically anthracene, are more prone to adsorption than straight chain, branched and cyclic aliphatic species. Titanium, aluminium and stainless steel show little adsorption ability and are suitable for larger-area applications. PTFE and Viton™ are suitable for use in small-area applications such as seals and filters. Nylon, polypropylene, polyethersulfone and cellulose acetate show stronger adsorption characteristics and should be avoided in the forms employed here. The ability of some materials to selectively adsorb organic compounds from solvent extracts can lower the sensitivity of life detection instruments. In future, it would be prudent to test all space instrument materials for their ability to adsorb target organic compounds from the solvent systems with which they will be used. This work has application beyond LMC and is relevant to future life detection instruments and their associated cleaning procedures that use solvents. The findings will also be pertinent to planned sample return missions involving caching and can inform the selection of materials used in sample return containers and during the subsequent processing of any returned samples.

Effects of Simulated Space Radiation on Immunoassay Components for Life-Detection Experiments in Planetary Exploration Missions

The Life Marker Chip (LMC) instrument is part of the proposed payload on the ESA ExoMars rover that is scheduled for launch in 2018. The LMC will use antibody-based assays to detect molecular signatures of life in samples obtained from the shallow subsurface of Mars. For the LMC antibodies, the ability to resist inactivation due to space particle radiation (both in transit and on the surface of Mars) will therefore be a prerequisite. The proton and neutron components of the mission radiation environment are those that are expected to have the dominant effect on the operation of the LMC. Modeling of the radiation environment for a mission to Mars led to the calculation of nominal mission fluences for proton and neutron radiation. Various combinations and multiples of these values were used to demonstrate the effects of radiation on antibody activity, primarily at the radiation levels envisaged for the ExoMars mission as well as at much higher levels. Five antibodies were freeze-dried in a variety of protective molecular matrices and were exposed to various radiation conditions generated at a cyclotron facility. After exposure, the antibodies' ability to bind to their respective antigens was assessed and found to be unaffected by ExoMars mission level radiation doses. These experiments indicated that the expected radiation environment of a Mars mission does not pose a significant risk to antibodies packaged in the form anticipated for the LMC instrument.

Development status of the life marker chip instrument for ExoMars

The Life Marker Chip (LMC) is one of the instruments being developed for possible flight on the 2018 ExoMars mission. The instrument uses solvents to extract organic compounds from samples of martian regolith and to transfer the extracts to dedicated detectors based around the use of antibodies. The scientific aims of the instrument are to detect organics in the form of biomarkers that might be associated with extinct life, extant life or abiotic sources of organics. The instrument relies on a novel surfactant-based solvent system and bespoke, commercial and research-developed antibodies against a number of distinct biomarkers or molecular types. The LMC comprises of a number of subsystems designed to accept up to four discrete samples of martian regolith or crushed rock, implement the solvent extraction, perform microfluidic-based multiplexed antibody-assays for biomarkers and other targets, optically detect the fluorescent output of the assays, control the internal instrument pressure and temperature, in addition to the associated instrument control electronics and software. The principle of operation, the design and the instrument development status as of December 2011 are reported here. The instrument principle can be extended to other configurations and missions as needed.

Extraction of polar and nonpolar biomarkers from the martian soil using aqueous surfactant solutions

The Life Marker Chip is being designed to detect the chemical evidence of life in the martian soil. It will use an aqueous surfactant solution to extract polar and nonpolar biomarkers from the martian soil and to transport them into an antibody-based detector for characterisation. Currently, a solution of 1.5gl−1 polysorbate 80 in 20:80 (vol:vol) methanol:water is being considered and appears to be suitable. Here, we have investigated the ability of a range of other surfactant solutions to extract a suite of eight standards spiked on the surfaces of the martian soil simulant JSC Mars-1 and tested the compatibility of the best two surfactants with a representative antibody assay for the detection of pyrene. The results show that using 20:80 (vol:vol) methanol:water as the solvent leads to increased recoveries of standards than using water alone. The poloxamer surfactants Pluronic® F-68 and Pluronic® F-108 are not effective at extracting the standards from JSC Mars-1 at any of the concentrations tested here. The fluorosurfactant Zonyl® FS-300 is able to extract the standards, but not as efficiently as polysorbate 80 solutions. Most successful of the alternative surfactants was the polysiloxane poly[dimethylsiloxane-co-[3-(2-(2-hydroxyethoxy)ethoxy)propyl]methylsiloxane] (PDMSHEPMS) which is able to extract the standards from JSC Mars-1 with an efficiency approximately equal to that of polysorbate 80 solutions of the same concentration. Enhanced recovery of the standards using polysorbate 80 and PDMSHEPMS solutions can be achieved by increasing the concentration of surfactant, from 1.5gl−1 to 10gl−1, leading to an increase in the recovery of standards of about 50%. Polysorbate 80 at concentrations of 1.5gl−1 and 10gl−1 and Zonyl® FS-300 and PDMSHEPMS (both at a concentration of 10gl−1) are also compatible with the representative pyrene antibody assay.

Planning for Mars Returned Sample Science: Final Report of the MSR End-to-End International Science Analysis Group (E2E-iSAG)

Returning samples from Mars to Earth for scientific analysis has been, and continues to be, among the highest priority objectives of planetary science. Partly for this reason, the 2011 Planetary Science Decadal Survey placed high priority on a proposed 2018 rover mission that would conduct careful in situ science and use that scientific information to select and cache samples that could be returned to Earth by a potential future mission. In order to ensure that the potential contributions of the 2018 rover to the proposed MSR Campaign are properly planned, this study was undertaken to consider the science of the MSR Campaign concept from end to end. This white paper is the principal output of the Mars Sample Return (MSR) End-to-End International Science Analysis Group (E2E-iSAG): a group chartered by MEPAG (Mars Exploration Program Analysis Group).

Immunological Detection of Small Organic Molecules in the Presence of Perchlorates: Relevance to the Life Marker Chip and Life Detection on Mars

The proposed ExoMars mission, due to launch in 2018, aims to look for evidence of extant and extinct life in martian rocks and regolith. Previous attempts to detect organic molecules of biological or abiotic origin on Mars have been unsuccessful, which may be attributable to destruction of these molecules by perchlorate salts during pyrolysis sample extraction techniques. Organic molecules can also be extracted and measured with solvent-based systems. The ExoMars payload includes the Life Marker Chip (LMC) instrument, capable of detecting biomarker molecules of extant and extinct Earth-like life in liquid extracts of martian samples with an antibody microarray assay. The aim of the work reported here was to investigate whether the presence of perchlorate salts, at levels similar to those at the NASA Phoenix landing site, would compromise the LMC extraction and detection method. To test this, we implemented an LMC-representative sample extraction process with an LMC-representative antibody assay and used these to extract and analyze a model sample that consisted of a Mars analog sample matrix (JSC Mars-1) spiked with a representative organic molecular target (pyrene, an example of abiotic meteoritic infall targets) in the presence of perchlorate salts. We found no significant change in immunoassay function when using pyrene standards with added perchlorate salts. When model samples spiked with perchlorate salts were subjected to an LMC-representative liquid extraction, immunoassays functioned in a liquid extract and detected extracted pyrene. For the same model sample matrix without perchlorate salts, we observed anomalous assay signals that coincided with yellow coloration of the extracts. This unexpected observation is being studied further. This initial study indicates that the presence of perchlorate salts, at levels similar to those detected at the NASA Phoenix landing site, is unlikely to prevent the LMC from extracting and detecting organic molecules from martian samples.

Synthesis and characterisation of immunogens for the production of antibodies against small hydrophobic molecules with biosignature properties

In the present study, five different classes of small hydrophobic molecular targets, atypical for antibody generation, were structurally modified in order to introduce suitable reactive functionalities and/or spacers which allow covalent coupling to a carrier protein resulting in a stable carrier–hapten complex. These targets were chosen to serve as markers of extant and/or extinct life in the context of the development of the Life Marker Chip (LMC), an antibody-based instrument, which is being developed by a UK-led international consortium for flight to Mars on board the joint ESA/NASA Mars exploration ExoMars mission. The hapten–protein conjugates were designed to be used as immunogens for antibody generation and immunoassay reagents in subsequent stages of the LMC development. The extent of protein modification due to covalent attachment of hapten was determined by two independent methods, i.e. trinitrobenzenesulfonic acid (TNBSA) titrations of remaining protein reactive groups and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of the resultant hapten–protein conjugates. In a further quality validation step, the conjugates were presented to an animal's immune system and polyclonal antibody titres with moderate specificity were obtained. These results suggest that conjugates synthesized as described herein can successfully be used in the generation of antibodies targeting small hydrophobic molecules.

New Solvents for Space Missions: Utility for Life Detection Instruments and Notable Terrestrial Applications

Instruments designed to test for signs of life on Mars must have operational simplicity and efficiency. One example is the Life Marker Chip being developed to fly on the forthcoming European Space Agency ExoMars mission. Target organic compounds include both polar and non polar molecules and, prior to our patented discovery, no solvent had been tested which effectively extracted both types of molecule in a fashion which was compatible with antibodybased detectors. We have compared the extraction efficiency of water-based solvents alongside conventional organic solvents to determine their suitability for extracting organic mixtures on space missions. Using a range of hydrocarbon standards and a Mars regolith simulant (JSC Mars-1) we have concluded that a water-methanol mix with 1.5 to 2.5 g/L of polysorbate 80 represents the most suitable solvent with extraction efficiencies that can achieve up to approximately 30% of that using conventional organic solvents (assuming 100% efficiency with 93:7 (vol:vol) dichloromethane:methanol mixtures). The surfactant solution will also provide solutions to terrestrial problems, one of which is explored in the patented work. Keywords: Life detection, organic compounds, extraction, Life Marker Chip, ExoMars, surfactants, Polysorbate, sonication, gas chromatograph, Spin-offs, polyoxyethylene

UK Mars research and priorities in the Aurora programme

John Bridges and Axel Hagermann summarize an RAS Special Discussion Meeting in January 2011, which looked at the prospects for the UK exploring Mars. High-resolution imaging and near-infrared mapping of the martian surface, together with detailed studies of martian meteorites and exobiology research, have revealed an increasing amount of detail about how climate and surface processes have changed over the history of Mars. The exploration of Mars is now being planned through a joint NASA–ESA programme. New missions such as the Mars Science Laboratory in 2012 and the Trace Gas Orbiter in 2016 will also improve our understanding about the evolution of Mars. Mission planning is being guided by recent reports of methane in the Mars atmosphere and the identification of minerals and landforms associated with water such as the Eberswalde Delta. Mars research is also acting as a spur for the development of mission concepts and analytical techniques such as new XRD techniques, microRaman and a Life Marker Chip. The second UK in the Aurora Programme meeting was held on 14 January 2011 at Burlington House, London. The aim was to bring together researchers, particularly in the field of Mars research and instrumentation, in order to provide a platform to show our research and to discuss what the priorities for future missions within Aurora should be. The Aurora programme encapsulates Europe's current plans, aiming to send unmanned spacecraft to explore our neighbouring world to examine its climate, search for evidence of past or present life, and learn how conditions there relate to those on Earth. The meeting was introduced by Frederick Taylor (Oxford). High-resolution imagery of some of the surface of Mars using the HiRise camera on the Mars Reconnaissance Orbiter (MRO) at 25 cm/pixel has changed our view of the evolution of its surface and climate. One of the ways our view of Mars has changed is the recognition of the influence of glacial and periglacial activity in shaping landscapes. Matt Balme (Open University) and Peter Gallagher (Trinity College Dublin) presented new observations of periglacial landforms that appear to have been formed by water as part of freeze–thaw cycles: self-organized patterns of stone stripes, polygons, circles and clastic solifluction lobes. The low density of impact craters suggests these terrains formed in a geologically recent era. Kate Goddard et al. (Imperial College) presented a study of youthful gullies and depositional fans on Mars in Mojave Crater. Understanding the role of water or other processes, including CO 2 ice sublimation, in the formation of such gullies is a crucial way of reaching a more accurate understanding of martian climate variations within the last few million years. Balme and Gallagher speculated that because perchlorates (identified at the Phoenix landing site) have eutectic temperatures below 240 K and can remain liquid at temperatures far below the freezing point of water, perchlorate brines may explain freeze–thaw processes and could cause geomorphological changes.

Novel solvent systems for in situ extraterrestrial sample analysis

The life marker chip (LMC) is being designed to test for the chemical signature of life in the soil and rocks of Mars. It will use an antibody array as part of its detection and characterisation system and aims to detect both polar and non-polar molecules at the sub-ppm to tens of ppb level. It is necessary to use a solvent to transfer organic compounds from the Martian samples to the LMC itself, but organic solvents such as dichloromethane or hexane, commonly used to dissolve non-polar molecules, are incompatible with the LMC antibodies. Hence, an aqueous-based solvent capable of dissolving the biomarkers that might exist in the soil or rocks of Mars is required. Solvent extractions of a Martian soil analogue, JSC Mars-1, spiked with a range of standards show that a 20:80 (vol:vol) mixture of methanol and water is incapable of extracting compounds insoluble in water. However, addition of 1.5 mg ml −1 of the surfactant polysorbate 80 produces extraction efficiencies of the aliphatic standards, hexadecane and phytane, equal to 25–30% of those produced by the common organic solvent mixture 93:7 (vol:vol) dichloromethane:methanol. Extraction of squalene and stigmasterol using the polysorbate solution is less efficient but still successful, at 5–10% of the efficiency of 93:7 dichloromethane:methanol. Such aliphatic compounds with occasional functional groups represent the compound classes to which most fossil organic biomarkers belong. The polysorbate solution did not extract the aromatic compounds pyrene and anthracene with great efficiency. A solvent of 20:80 methanol:water with 1.5 mg ml −1 polysorbate 80 is therefore capable of selectively extracting aliphatic biomarkers from Martian samples and transferring them to the antibody sites on the life marker chip.

Searching for Life on Mars: Selection of Molecular Targets for ESA's Aurora ExoMars Mission

The European Space Agency's ExoMars mission will seek evidence of organic compounds of biological and non-biological origin at the martian surface. One of the instruments in the Pasteur payload may be a Life Marker Chip that utilizes an immunoassay approach to detect specific organic molecules or classes of molecules. Therefore, it is necessary to define and prioritize specific molecular targets for antibody development. Target compounds have been selected to represent meteoritic input, fossil organic matter, extant (living, recently dead) organic matter, and contamination. Once organic molecules are detected on Mars, further information is likely to derive from the detailed distribution of compounds rather than from single molecular identification. This will include concentration gradients beneath the surface and gradients from generic to specific compounds. The choice of biomarkers is informed by terrestrial biology but is wide ranging, and nonterrestrial biology may be evident from unexpected molecular distributions. One of the most important requirements is to sample where irradiation and oxidation are minimized, either by drilling or by using naturally excavated exposures. Analyzing regolith samples will allow for the search of both extant and fossil biomarkers, but sequential extraction would be required to optimize the analysis of each of these in turn.

Automated payload and instruments for astrobiology research developed and studied by German medium-sized space industry in cooperation with European academia

For more than a decade Kayser-Threde, a medium-sized enterprise of the German space industry, has been involved in astrobiology research in partnership with a variety of scientific institutes from all over Europe. Previous projects include exobiology research platforms in low Earth orbit on retrievable carriers and onboard the Space Station. More recently, exobiology payloads for in situ experimentation on Mars have been studied by Kayser-Threde under ESA contracts, specifically the ExoMars Pasteur Payload. These studies included work on a sample preparation and distribution systems for Martian rock/regolith samples, instrument concepts such as Raman spectroscopy and a Life Marker Chip, advanced microscope systems as well as robotic tools for astrobiology missions. The status of the funded technical studies and major results are presented. The reported industrial work was funded by ESA and the German Aerospace Center (DLR).

The specific molecular identification of life experiment ( SMILE)

We describe a compact, highly integrated instrument concept for the detection and identification of a wide range of molecules associated with extinct/extant life or potential life processes. The Specific Molecular Identification of Life Experiment ( SMILE) will be sensitive to the presence of a range of target molecules using both electrical and optical transduction techniques, and incorporates molecular imprinted polymers in addition to traditional biological receptors such as antibodies. A number of versions of the concept are possible depending on available resources e.g. mass, volume, etc. The full concept utilises a novel imaging interferometer where a large number of molecular receptors are deposited on the measurement plane of an imaging interferometer and read out by an imaging detector, enabling multiple targets—biomarkers—within a sample to be measured simultaneously. The optics can also form the basis of an UV–NIR imaging Fourier spectrometer allowing basic mineralogy studies to be conducted using optical properties to assist in the determination of the geological context of the samples. By incorporating micro-fabricated transducer arrays, micro-fluidics and artificial molecular recognition systems, as well as recombinant antibody technology with appropriate integration methods, SMILE forms a compact and robust “Life Marker Chip” which has been proposed for future planetary missions including ESA's ExoMars mission, where the instrument offers the possibility of conducting a direct in situ search for signs of past or present biological activity on Mars. In addition to its role in planetary exploration, derivatives of SMILE have multiple terrestrial applications in fields such as forensic analysis and environmental monitoring.