Detecting Nonvolatile Life- and Nonlife-Derived Organics in a Carbonaceous Chondrite Analogue with a New Multiplex Immunoassay and Its Relevance for Planetary Exploration

Abstract Potential martian molecular targets include those supplied by meteoritic carbonaceous chondrites such as amino acids and polycyclic aromatic hydrocarbons and true biomarkers stemming from any hypothetical martian biota (organic architectures that can be directly related to once-living organisms). Heat extraction and pyrolysis-based methods currently used in planetary exploration are highly aggressive and very often modify the target molecules, making their identification a cumbersome task. We have developed and validated a mild, nondestructive, multiplex inhibitory microarray immunoassay and demonstrated its implementation in the SOLID (Signs of Life Detector) instrument for simultaneous detection of several nonvolatile life- and nonlife-derived organic molecules relevant in planetary exploration and environmental monitoring. By utilizing a set of highly specific antibodies that recognize D- or L-aromatic amino acids (Phe, Tyr, Trp), benzo[a]pyrene (B[a]P), pentachlorophenol, and sulfone-containing aromatic compounds, respectively, the assay was validated in the SOLID instrument for the analysis of carbon-rich samples used as analogues of the organic material in carbonaceous chondrites or even Mars samples. Most of the antibodies enabled sensitivities at the 1–10 ppb level and some even at the part-per-trillion level. The multiplex immunoassay allowed the detection of B[a]P as well as aromatic sulfones in a water/methanol extract of an Early Cretaceous lignite sample (ca. 140 Ma) representing type IV kerogen. No L- or D-aromatic amino acids were detected, reflecting the advanced diagenetic stage and the fossil nature of the sample. The results demonstrate the ability of the liquid extraction by ultrasonication and the versatility of the multiplex inhibitory immunoassays in the SOLID instrument to discriminate between organic matter derived from life and nonlife processes, an essential step toward life detection outside Earth.

[1]  A. Yingst,et al.  A Habitable Fluvio-Lacustrine Environment at Yellowknife Bay, Gale Crater, Mars , 2014, Science.

[2]  P. Rutkowski,et al.  Identification of Organic Sulfur Compounds in Supercritical Extracts from Polish Lignite , 2001 .

[3]  D. Ming,et al.  Volatile and Organic Compositions of Sedimentary Rocks in Yellowknife Bay, Gale Crater, Mars , 2014, Science.

[4]  Gary Ruvkun,et al.  Radiation resistance of biological reagents for in situ life detection. , 2013, Astrobiology.

[5]  Javier Gómez-Elvira,et al.  A microbial oasis in the hypersaline Atacama subsurface discovered by a life detector chip: implications for the search for life on Mars. , 2011, Astrobiology.

[6]  M. Wilchek,et al.  ANTIBODIES CAN RECOGNIZE THE CHIRAL CENTER OF FREE ALPHA -AMINO ACIDS , 1998 .

[7]  George G Klee,et al.  Antibody-based protein multiplex platforms: technical and operational challenges. , 2010, Clinical chemistry.

[8]  Xianyong Wei,et al.  Sulfur-containing species in the extraction residue from Xianfeng lignite characterized by X-ray photoelectron spectrometry and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry , 2015 .

[9]  Javier Gómez-Elvira,et al.  SOLID2: an antibody array-based life-detector instrument in a Mars Drilling Simulation Experiment (MARTE). , 2008, Astrobiology.

[10]  Michel Maurette,et al.  A Search for Extraterrestrial Amino Acids in Carbonaceous Antarctic Micrometeorites , 1998, Origins of life and evolution of the biosphere.

[11]  Christopher P. McKay,et al.  Reanalysis of the Viking results suggests perchlorate and organics at midlatitudes on Mars , 2010 .

[12]  Ian Wright,et al.  Investigating the variations in carbon and nitrogen isotopes in carbonaceous chondrites , 2003 .

[13]  V. Parro,et al.  Multiplex Fluorescent Antibody Microarrays and Antibody Graphs for Microbial and Biomarker Detection in the Environment , 2015 .

[14]  M. Sephton,et al.  Organic geochemistry of late Jurassic paleosols (Dirt Beds) of Dorset, UK , 2012 .

[15]  Zita Martins,et al.  Type IV kerogens as analogues for organic macromolecular materials in aqueously altered carbonaceous chondrites. , 2013, Astrobiology.

[16]  R. Niessner,et al.  Rapid and simultaneous detection of ricin, staphylococcal enterotoxin B and saxitoxin by chemiluminescence-based microarray immunoassay. , 2014, The Analyst.

[17]  Stuart L. Schreiber,et al.  Printing Small Molecules as Microarrays and Detecting Protein−Ligand Interactions en Masse , 1999 .

[19]  Javier Gómez-Elvira,et al.  Instrument development to search for biomarkers on mars: Terrestrial acidophile, iron-powered chemolithoautotrophic communities as model systems , 2005 .

[20]  Jared A. Carter,et al.  A label-free, multiplex competitive assay for small molecule pollutants. , 2016, Biosensors & bioelectronics.

[21]  Javier Gómez-Elvira,et al.  SOLID3: a multiplex antibody microarray-based optical sensor instrument for in situ life detection in planetary exploration. , 2011, Astrobiology.

[22]  Javier Gómez-Elvira,et al.  A multi-array competitive immunoassay for the detection of broad-range molecular size organic compounds relevant for astrobiology , 2006 .

[23]  D. Cullen,et al.  Immunological detection of small organic molecules in the presence of perchlorates: relevance to the life marker chip and life detection on Mars. , 2011, Astrobiology.

[24]  Andrew Steele,et al.  Searching for life on Mars: selection of molecular targets for ESA's aurora ExoMars mission. , 2007, Astrobiology.

[25]  J. Rullkötter,et al.  The structure of kerogen and related materials. A review of recent progress and future trends , 1990 .

[26]  Damià Barceló,et al.  Biosensors as useful tools for environmental analysis and monitoring , 2006, Analytical and bioanalytical chemistry.

[27]  Á. Maquieira,et al.  Development of an enzyme-linked immunosorbent assay for pentachlorophenol , 2002 .

[28]  P. Moretto,et al.  Investigation of low-energy proton effects on aptamer performance for astrobiological applications. , 2011, Astrobiology.

[29]  Suxia Zhang,et al.  Monoclonal antibodies with group specificity toward sulfonamides: selection of hapten and antibody selectivity , 2013, Analytical and Bioanalytical Chemistry.

[30]  María-José Bañuls,et al.  Development of hapten-linked microimmunoassays on polycarbonate discs. , 2010, Analytical chemistry.

[31]  Shuo Wang,et al.  Review on enzyme-linked immunosorbent assays for sulfonamide residues in edible animal products. , 2009, Journal of immunological methods.

[32]  Danni Li,et al.  A microfluidic multiplex proteomic immunoassay device for translational research , 2015, Clinical Proteomics.

[33]  J. Watson,et al.  Sulfate Minerals: A Problem for the Detection of Organic Compounds on Mars? , 2015, Astrobiology.

[34]  Ángel Maquieira,et al.  Analysis of Atrazine in Water and Vegetables Using Immunosensors Working in Organic Media , 2003 .

[35]  Mark A. Sephton,et al.  Macromolecular organic materials in carbonaceous chondrites: A review of their sources and their role in the origin of life on the early earth , 2000 .

[36]  P Coll,et al.  Organic molecules in the Sheepbed Mudstone, Gale Crater, Mars , 2015, Journal of geophysical research. Planets.

[37]  R. Philp,et al.  Pyrolysis-GC analyses of the recent cyanobacterial mats reacted with H2S under mild condition. , 1995 .

[38]  Reinhard Niessner,et al.  Development of a highly sensitive monoclonal antibody based ELISA for detection of benzo[a]pyrene in potable water. , 2005, The Analyst.

[39]  D P Glavin,et al.  Extraterrestrial amino acids in Orgueil and Ivuna: Tracing the parent body of CI type carbonaceous chondrites , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[40]  Everett Shock,et al.  The organic composition of carbonaceous meteorites: the evolutionary story ahead of biochemistry. , 2010, Cold Spring Harbor perspectives in biology.

[41]  S. Larter,et al.  Improved kerogen typing for petroleum source rock analysis , 1985, Nature.

[42]  D. Cullen,et al.  Effects of simulated space radiation on immunoassay components for life-detection experiments in planetary exploration missions. , 2012, Astrobiology.

[43]  Oliver Hofstetter,et al.  Antibody-based multiplex analysis of structurally closely related chiral molecules. , 2011, The Analyst.

[44]  A. Burton,et al.  Understanding prebiotic chemistry through the analysis of extraterrestrial amino acids and nucleobases in meteorites. , 2012, Chemical Society reviews.

[45]  Stephen Killops,et al.  An introduction to organic geochemistry , 1993 .

[46]  J. Rodriguez-Manfredi,et al.  Prokaryotic communities and operating metabolisms in the surface and the permafrost of Deception Island (Antarctica). , 2012, Environmental microbiology.

[47]  K. Biemann,et al.  The implications and limitations of the findings of the Viking organic analysis experiment , 1979, Journal of Molecular Evolution.

[48]  B. Tissot,et al.  Influence of Nature and Diagenesis of Organic Matter in Formation of Petroleum , 1974 .

[49]  J. Holt,et al.  Development status of the life marker chip instrument for ExoMars , 2012 .

[50]  J. W. Pickering,et al.  A multiplexed fluorescent microsphere immunoassay for antibodies to pneumococcal capsular polysaccharides. , 2002, American journal of clinical pathology.

[51]  M. Sephton,et al.  Extraction of polar and nonpolar biomarkers from the martian soil using aqueous surfactant solutions , 2012 .

[52]  V. Parro,et al.  Assessing antibody microarrays for space missions: effect of long-term storage, gamma radiation, and temperature shifts on printed and fluorescently labeled antibodies. , 2011, Astrobiology.

[53]  An Optical Immunosensor for Pesticide Determination in Natural Waters , 2006 .

[54]  Á. Maquieira,et al.  Development of a group-specific immunoassay for sulfonamides. Application to bee honey analysis. , 2007, Talanta.