Antibody microarrays: molecular recognition for biomarker detection

Biosensors based on the specificity of biological recognition (bio-affinity) have been extensively developed for biomarker detection (nucleic acids, proteins, lipids, other metabolites). DNA and protein micro and nanotechnologies allow covalent binding of thousands of probes in a small area on a solid support (plastic, glass, micro and nano-spheres, etc). Smaller reaction volumes and higher reaction kinetics, together with the great potential for miniaturization and robotizing, makes microarray technology a powerful system for in situ analysis of biomarkers in astrobiology as well as environmental monitoring.

Molecular recognition by bio-affinity systems, such as the highly specific reaction between antigens and antibodies, protein receptors and ligands, or the complementary between single-stranded nucleic acids, have been extensively used in biosensor industry. In fact, the most employed biosensors for environmental applications used antibodies, due to the availability of many of them against specific contaminants. There are thousands of commercial antibodies raised against compounds ranging from small molecules such as amino acids, sugars, and lipids, to large polymers and whole cells.

Antibodies for planetary exploration

Antibodies are large glycoproteins that can be chemically modified (by other substances, like fluorochromes, other proteins, or even other antibodies), without affecting the binding capacity and specificity of the antibody to its antigen. Apart from antibodies there are other types of specific capturing molecules: lectins, a group of nonenzymatic proteins with high specificities and affinities for mono and oligosaccharides; affybodies, in vitro selected binding proteins; aptamers, in vitro selected binding oligonucleotides; or the molecularly imprinted polymers (MIPs), organic polymers formed in the presence of a molecule that is extracted afterwards, thus leaving complementary cavities behind.

Antibody (or other bio-affinity capturing molecules) arrays are excellent complementary techniques to others like GC-MS or capillary electrophoresis, for planetary exploration. Some important strengths of the antibody microarrays are:

  • Multiple parallel assays at a time can be run simultaneously.
  • The presence of hundreds of molecules can be tested in one assay.
  • Broad range of molecular sizes can be detected, from aa size to cells.
  • No special external calibration or standards required for the assay.
  • Many negative and positive controls can be incorporated and analyzed at the same time as samples.
  • Sensitivity from ppb to ppt.
  • Results are very easy to analyze. Putative biomarkers are identified due to the specificity of the probes and their predetermined location. Antibodies only react with those molecules containing epitopes with three-dimensional structures equal or highly similar to those used to produce the antibodies, which means that it is possible to get unambiguous results.

Stability of the antibodies for planetary exploration

Antibodies can be high stable macromolecules when they are kept under appropriate conditions. Several parameters like the nature of the solvent, temperature, lyophilization, freezing, salts, light, or grafting step can affect the three-dimensional structure of antibodies and consequently the affinity for their antigens. Apart of that, planetary exploration requires functional antibodies under deeply penetrating low energy radiation, microgravity, temperature cycles and its variations, etc. The pharmaceutical industry has developed methods for antibody stability and it is well known that most antibodies maintain their activity for years when frozen (between –20 ºC and –80 ºC) in a saline solution or lyophilized and kept at ambient temperature protected from light. Usually stabilizers such as sugars/polyols are added to reduce the degradation of active components during processing and storage. Lyophilization in the presence of sugars (sucrose or trehalose) is commonly used to provide long-term storage stability to protein pharmaceuticals. It is believed that proteins reach a highly viscous amorphous glassy state with low molecular mobility and low reactivity. Under such state the stability is highly dependent on storage temperature. Storage at temperatures above the glass transition temperature (usually ca. 50 ºC) the glassy material become less viscous, the molecular mobility increases and the stability decreases.

Antibodies can also be preserved by drying at ambient temperature and at atmospheric pressure in the presence of trehalose or other formulations. We have experimentally determined that printed antibody arrays on microscope slides are stable (keeping more than 90 % of their functionality when compared to time zero) for more than 9 months stored at room temperature, even at 37 ºC in the presence of stabilizing solutions developed by CAB and by the company Biotools S.A. (De Diego-Castilla et al., 2011). Fluorescently labeled and lyophilized antibodies are stable over more than 50 temperature cycles (24 h each) of –20 to +50 ºC, and even after an exposure of 150 Gy  of high penetrating low energy radiation (De Diego-Castilla et al., 2011) (Gy=gray. One gray is the absorption of one joule of radiation energy by one kilogram of matter). In agreement with our results, Thompson et al., (2006) have reported that no significant alteration in the absorption and emission wavelengths or the quantum yields of two fluorescent dyes was found after a proton and helium ion radiation. Maule et al. (2003) showed that the level of an antibody-antigen binding did not differ between microgravity, Martian gravity and Earth’s gravity conditions.


Molecular biomarker targets for antibody production

Our hypothesis assume that if life ever existed on Mars or elsewhere, the organisms dealing with the Martian environmental conditions must contain similar molecular mechanisms that those living under similar conditions on Earth. Consequently, our strategy for testing the presence of life or its remains consists in the production of antibodies against whole microorganisms, specific components or biochemicals obtained from terrestrial analog environments to Mars, to biochemical compounds which are common to many different types of microorganisms (nucleotides, sugars, aminoacids, vitamins and coenzymes, etc), or other well evolutionary conserved macromolecules like some proteins, polysaccharides, or polynucleotide sequences. Many of these molecules can be considered good molecular biomarkers and their detection somewhere would be indicative of the actual or recent live (Parro et al., 2008). We have produced, tested and validate an antibody microarray containing more than 300 antibodies for both environmental and astrobiology applications (Rivas et al. 2008; Parro et al., 2011).

Terrestrial analogues

On Earth, It is possible to find life in practically all those places where one can find liquid water. And many times liquid water or traces of it occurs in places known as extreme environments. These are created because of the permanent existence of extreme values of certain physico-chemical parameters like, temperature, pressure, pH, redox potential, radiation, water content, salinity, etc. From a terrestrial point of view, Mars and the Jupiter’s satellite Europa are extreme environments where several or all of the mentioned parameters seem to be very far from “normal” values in many places of the planet. One of the main approaches to study the possibility and the possible features of extraterrestrial life is to study life in extreme conditions on Earth. Thus, the study of several terrestrial analogues for certain Mars environments can serve as useful models at least to understand the main features of martian life if ever existed. We mainly focus on three environments that could share many physico-chemical characteristics to others thought to exist on Mars: Hydrothermal, acid and iron rich, and permafrost. The idea of subsurface environments for extant biology is strengthened by evidence suggestive of hydrothermal activity on Mars in the relatively recent past. However, whether Mars is still geologically active is not yet determined. There may well be subsurface regions where liquid water is available, and where the local conditions might support the growth of an indigenous biota. If microbial life ever existed on Mars, probably iron metabolism played a central role both as energy source and for the formation of structural components (metalloproteins). Earth Polar Regions are excellent analogues to many places of Mars surface because both are subjected to similar stresses. This is the case of the Antarctic Dry Walleys, extremely cold and dry places, with very low microbial and organic content.

Acidic, metal-rich, highly oxidant environments

Both MarsExpress (by ESA) and MER (Mars Exploration Rovers, by NASA), have reported results that link with the presence of water in the primitive Mars. The obtained data indicate that several large regions of Mars were covered with water, as well as the MER in the Meridiani Planum provided us with evidences of the presence of minerals that can only be formed in presence aqueous environment. There were arguments enough to think that the water belonged to an ancestral acidic ocean. Recently, the OMEGA (“Observatoire pour la Minéralogie, l’Eau, les Glaces et l’Activité”) instrument of the Mars Express probe confirmed the existence of a large and representative aqueous-sulphated systems like in the Meridiani Planum. The acidity might be the reason why large amounts of carbonates have not been detected yet since these chemical conditions are not suitable for its production. As explained in a recent model, the SO2 erupted from volcanoes should play a role like the CO2 on Earth by creating a greenhouse effect which allow the water to remain liquid (acidic oceans) for hundred of years. Thus, it is possible to accumulate sulphates (MER detection) instead of carbonates. It is very interesting for the planetary exploration science to study the terrestrial extreme environments that resemble hypothetical planetary ecosystems. Thereby the Tinto’s deposits of the Holocene and Plio-Pleistocene epochs are similar to the sulphates and hematite rich sedimentary rocks discovered by the Opportunity rover in the Meridiani Planum.

Terrestrial chemolithoautotrophs organisms obtain energy by the oxidation of electron donating inorganic reduced molecules in their environments. The acidophilic (pH 0.8-2.3) microorganisms living in the world’s largest pyrite strip (located in the Iberian Peninsula, Tinto River basin), like Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans, have a minimal nutritional requirements. They only need Fe2+ as source of energy, CO2, N2 and O2 as source of carbon, nitrogen and electron acceptor respectively, a part of water and mineral salts to make their metabolism runs. Moreover, A. ferrooxidans has a versatile behaviour while growing in presence of Fe2+/O2 or H2/O2 (aerobic conditions; Fe2+ and H2, electron donors; O2 electron acceptor) as well as in presence of H2/Fe3+, H2/SO, or SO/Fe3+ (anaerobic conditions; SO and H2, electron donors; Fe3 y SO electron acceptors). Although L. ferrooxidans is a strict aerobic bacterium, it is capable of surviving in anaerobic conditions over long periods of time and it exhibits at high metabolic rate when the level of oxygen is lower than 15%. Moreover, we reported by gene expression studies with L. ferrooxidans that these bacteria are in fact true polyextremophiles, that is, they can grow under several extreme conditions: low pH (1.8), high metal content (20 gL-1 of Iron), high salt content (>80 g L-1 of SO4=), or high oxidative stress.

Hypothetically, if the acidic oceans and CO2 and SO2 gases existed on the primitive Mars, they could have provided microorganisms with: Iron as energy source, CO2 as carbon source, N2 as nitrogen source, and sulphured compounds as energy source for sulphur-reducer bacteria. Moreover, it has been reported that the predicted amount of N2 in the primitive Martian atmosphere could have been high enough to allow microorganisms like Rhodobacter y Mesorhyzobium sp. to fix the nitrogen. On the other hand, although we lack of data to clarify the origin, the traces of methane that have been detected in the Martian atmosphere are thought to be released by the hypothetical methanogenic bacteria in the subsoil. Therefore, the pyritic strip in the Iberian Peninsula still constitutes a splendid model since it holds methanogenic activity underground as well as in several zones of the Tinto’s river. Nowadays the scientific community hopes to find signs of past or present life related to microorganisms which lived in the subsoil since the radiations above underground are believed incompatibles with life as it is known on Earth.

Hydrothermal environments

Deep sea hydrothermal systems were only discovered on Earth in the late 1970s but are now known to represent one of the major pathways for transfer of heat in our planet’s interior as well as representing one of the major chemical transport mechanisms to the oceans. Of particular interest are the chemosynthetic biological energy cycles around the hydrothermal systems, which have implications for the origin of life itself. Methods of chemical analysis in these remote environments have, by necessity, depended on collecting samples at depth which are then transferred to a surface ship and/or land-based for analysis. This is far from ideal, because of subjection of the sample to exceptionally large changes in pressure, temperature, oxygenation, etc prior to analysis. On the other hand, this oceanographic dilemma reduces the dignity of problems of remote sampling control on other planets to a problem of the size order more than being an obstacle as such. The long-term goal is on Earth, as well, to replace laboratory analyses by in situ monitoring as far as possible. The most easily accessible source of energy for micro-organisms in the lithosphere of terrestrial planets is the molecular hydrogen (H2) that is formed during oxidation of Fe(II) inherent in minerals. One of the most easily weathered Fe(II) minerals is olivine. This is a solid solution of the Mg mineral forsterite (Mg2SiO4) with a minor contribution (10-20%) of the Fe(II) mineral fayalite (Fe2SiO4) Olivine 45% (SiO2) of the upper part

Icy environments

Many photosynthetic cyanobacteria and algae from the Polar Regions are subjected to great environmental extremes and they synthesize biomolecules to cope with these conditions. These extremes include low temperatures, extreme temperature variations, high solar radiation and periods of desiccation. Polysaccharides, for instance, are synthesized to deal with desiccation and compounds such as scytonemin are synthesized to deal with UV radiation stress. The stresses to be found on Mars are similar to those found in the polar regions of the Earth. Thus, organisms from Polar Regions provide excellent model systems for understanding survival responses of any putative biota to Martian conditions and may provide insights into the types of biomolecules produced by micro-organisms in response to Martian environmental stresses. For example, Chroococcidiopsis spp, an extremophilic cyanobacterium, exists naturally in the endolithic habitat in both hot and cold deserts of the world, being found as coherent bands of organisms growing in the near-surface environment of the rocks. This genus is resistant to desiccation and even ionizing radiation, the latter extreme also being important for organisms on the surface of Mars that will be exposed to a much higher ionizing radiation flux than they would be on Earth.

Extremely Dry Deserts

For years, the Atacama desert has been used by different research groups within NASA as a Mars analogue. The Atacama desert is one of the most accurate terrestrial analogues for Mars environments because it combines the formation of two key inorganic compounds: chlorides and perchlorates. Similar to events on Mars, harsh arid conditions promoted an extreme oversaturation of the ground and surface water solutions that resulted in nearly-exclusive precipitation of halite, the end-member in evaporation from brines, with no other mineral phase. The bounding regions of the Salar Grande (Cordillera de la Costa, región de Atacama, Chile) are characterized by saline subsoils associated with nitrate deposits containing chlorides, sulfates, chlorates, chromates, iodates, and perchlorates.  During July 2009 we performed an astrobiological field campaign, “AtacaMars2009,” where we tested SOLID3 and LDChip300 and study the geomicrobiology of the Atacama subsurface at the west side of the Salar Grande (Parro et al., 2011).