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Sensitive amino acid composition and chirality analysis with the Mars Organic Analyzer (MOA) Alison M. Skelley
Multi-Layered Microfluidic Devices for Amino Acid Analysis:
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Searching for Evidence of Life on Mars
Jeffrey L. Bada
European Space Agency Exploration Seminar Series
Noordwijk, Holland
November 12, 2004
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Microfabricated Organic Analyzer (MOA) for in situ Exploration of Mars
and other Solar Bodies
ASTEP NASA investigators meeting, University of Colorado at Boulder
January 20-21, 2004
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What are the best ways to look for extinct or extant
life on Mars? Thinking outside the box
American Geophysical Union Annual Meeting
San Francisco, CA
December 15–19, 2003
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Amino Acid Composition and Chirality Analysis in Microfabricated Devices
American Geophysical Union Annual Meeting
San Francisco, CA
December 15–19, 2003
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AstroBioLab:
A Mobile Biotic And Soil Analysis Laboratory
Third European Workshop on Exo-Astrobiology, Madrid, Spain
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Microfabrication Technologies for PCR-CE and Amino Acid Analysis
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ASTROBIOLOGY 101
An undergraduate research project by Dmitri Terterov
General Related FAQ’s:
How do we define life?
Life is broadly defined as an organized structure that is able to reproduce forming like structures, and that is able to produce, convert, and utilize energy for growth and reproduction by interacting with its environment.
How do we think life originated on earth?
We believe that some of the most basic organic molecules got synthesized spontaneously under certain, early-Earth conditions. Such basic molecules slowly concentrated on hot rocks and began to react to form larger, more complex organic molecules. Congregations of such molecules got trapped in membranes and slowly increased in complexity until they resembled the first cell, which could reproduce and carry out basic metabolic activity.
What is some experimental basis for this origin hypothesis?
In the 1950’s Miller and Urey simulated the hypothetical, reducing atmosphere of the early Earth. This “atmosphere” included dense water vapor, methane, ammonia, and hydrogen. When this mixture of gasses was subjected to an electric spark (i.e. a lightning bolt), a number of organic molecules formed spontaneously, some of which were amino acids.
Based on this origin hypothesis what are the requirements for life?
First and foremost, conditions have to be such that chemical reactions can occur at a relatively high rate. The minimum requirement for this is a “moderate” temperature. A temperature that is too low makes many chemical reactions occur at too slow a rate or not at all. At temperatures that are too high, complex biopolymers break down to their basic components; no complex molecules can survive in such conditions. The second important condition is the availability of a good solvent because most bioorganic processes occur in solution. Water, with its unique properties, seems to be the ideal solvent (although probably not the only possibility). Its wide solvent range, 0-100°C, makes it available in liquid phase on a relatively broad range of temperatures. For more information, follow this link to a discussion on the necessity of water featuring Jeff Bada: http://www.pbs.org/wgbh/nova/mars/essential.html.Why is Mars a good place to start our search for life in the universe?
Mars is one of the planets in the Solar System that falls within the life-favoring range of temperatures described above. Moreover, there is current evidence for the presence of water (only as ice so far) on Mars as well as that for the past presence of liquid water. There are also some arguments for the presence of amino acids in Martian soil. These arguments are based on the analysis of the meteorite ALH84001 which was established to have its origin on Mars. Certain amino acids have been detected in this meteorite, although the hypothesis that terrestrial contamination is the source of these amino acids has not yet been rejected (for more information on this see A Search for Endogenous Amino Acids in Martian Meteorite ALH84001) link to http://astrobiology.berkeley.edu/PDFs articles/Bada_science1998.pdf. Finally, an obvious reason is that Mars is one of our two neighbor-planets and therefore is easiest to reach.
Current Project – Search for the Extinct or Extant Life on Mars: The Theory Behind It
How can chirality be used as an indirect indicator of the presence of life?
Many organic molecules are chiral. Although the difference among the stereoisomers seems to be really subtle, only one of the stereoisomers is usually functional in terms of its biological role in an organism. Therefore, the synthesis mechanisms that have evolved in living organisms are such that they process only one of the two possible optical isomers. This direction of thought ties the chirality of a sample of organic molecules to past or present existence of life.
What conclusions could we make if we had a way to analyze a sample of organic molecules for chirality?
If the sample is found to be racemic , this observation indicates that it was synthesized abiotically. Organic molecules formed in the Miller-Urey experiment were racemic (see description above) because there was an equal probability of forming one isomer or the other. If, on the other hand, the results of the analysis indicate that the sample is homochiral, the only conclusion that follows is that these organic molecules were synthesized by an organism in a fashion that allowed only for one stereoisomer.
What happens to chirality of a mixture of organic molecules over time?
A homochiral mixture does racemize (become racemic ) over time, however the process is slow for certain organic molecules, under certain conditions. Therefore, no chiral analysis will yield a perfectly homochiral result unless the sample is obtained from a living or a “freshly dead” organism. Instead, the result will most likely be something intermediate between homochirality and racemity. This still means that the molecules do have a biotic origin, but far back in time. Putting information about the rates of racemizeation of a specific molecule in a specific environment together with the data of the actual chiral composition of a sample makes it possible to estimate how long ago the racemization began – how long ago the organism (the source of these molecules) died. This, of course, would only be a rough estimate because the sample might have originated from many different organisms that died at different times. However, it could give us an idea of how long ago life existed on Mars.
Why use Amino Acids as the target organic molecules for the search of life on Mars?
First of all, amino acids racemize very slowly under the conditions on Mars, which makes it possible to “look” far back in time using the chirality arguments. The dry, cold Martian environment sets racemization times of amino acids on the order of 10,000 years. Secondly, since amino acids were produced in the Urey-Miller synthesis (see description above) their presence on Mars is a reasonable assumption.
Microfabricated Organic Analyzer (MOA) FAQ’s:
What does MOA do?
It performs an analysis of a mixture of organic molecules, detecting/ recognizing specific amino acids and separating them according to chirality. In other words, it is able to tell whether the amino acids found are racemic or homochiral – whether they have an abiotic or a biotic origin, as well as which amino acids are present.
Where does it do it?
This whole analysis is performed right on the site – on Mars. In situ analysis is advantageous because there are numerous technical and financial difficulties associated with bringing a sample back and analyzing it here on Earth. Even more importantly, transporting the sample greatly increases the chances of its contamination.
What general method does MOA use?
MOA uses a variation of electrophoresis called capillary electrophoresis. This involves the application of an electric field to a capillary filled with a running buffer and results in a separation based on size and charge of the migrating molecules.
What modifications have been made to the general method in order to make MOA compatible with extra-terrestrial in situ exploration?
The whole system that is required for capillary electrophoresis, including the separation channel, pumps (used to move the running buffer around), sample storage/waste containers, etc., has been microfabricated and placed on a 4 mm thick “chip” only 10 cm across. Moreover, all the steps required for a successful separation were automated and put under the control of a computer. Since the volumes processed are on the order of nanoliters to microliters and the level of automation is high, an average separation takes less than 5 minutes. This means that multiple samples can be analyzed in order to improve statistical significance. Thus, the usual difficulties associated with extraterrestrial in situ exploration such as the size and weight of the apparatus and the speed of sample processing and analysis have been overcome.
Why are chiral separations difficult to achieve?
Chiral separations are usually difficult to achieve because the different enantiomers have identical physical and chemical properties. They have the same boiling/freezing points, same solubilities, same mobility in a separation channel, same acidity, etc. Performing a chiral separation is analogous to trying to separate right hand gloves from left hand gloves in the dark. They are all the same size, all made out of the same material, all reflect light the same way, and have the same shape. Such a task may seem impossible at a first glance.
Using what general principle are chiral separations achieved?
Continuing the analogy of sorting out the gloves, the problem of chiral separation of the gloves can be solved by using one’s hand as a temporary template. The separation process would then proceed as follows: the person trying to sort out the gloves tries on the gloves one by one. Once a glove is on the hand the feeling of whether it fits or not is definite. The person then takes off the glove and puts it in the appropriate pile. Thus, the hand in this example serves as an indicator that gives a different result based on the chirality of the glove.
How does MOA achieve chiral separations of the amino acids?
Capillary electrophoresis is used as the general method, but there is one major modification. A solution of a molecule called HydoxypropylßCyclodextrin (HPbCD) is added to the buffer solution (HPßCD is chiral itself). The molecule’s bucket-like structure makes it possible for the side chain of an amino acid to fit inside tightly with the rest of the molecule sticking out of the cavity. Such a HPßCD+amino acid complex is obviously much larger than the amino acid by itself (has a higher drag) and thus travels slower through the solution when a voltage is applied. However, since HPßDC has a chirality of its own, it interacts with one of the amino acid forms more than with the other. In other words, one of the enantiomers of the amino acid spends (on average) more time inside the cavity and thus on average has a lower speed through the channel.
Here is a schematic illustrating such a separation in the electrophoretic capillary (also see capillary electrophoresis):
What is the detection method used once the separation has been achieved?
Fluorescence spectroscopy is used to detect the amino acids. Since the amino acids are not fluorescent themselves, fluorescamine is used as the labeling agent. When excited (hit) with a laser beam, it emits light. This emitted light is detected.
How is MOA related to the previously developed - MOD (Mars Organic Detector)?
MOA is an enhanced version of MOD, coupling the automated lab-on-chip system (for identification of amino acids and chiral separations) to the MOD platform. MOD isolates and detects amino acids in Martian soil, but it is not able to analyze the different amino acids or their chiralities. The MOD platform, however, is an important component because it is responsible for the isolation of the sample from the Martian soil and for amino acid labeling. In essence the MOD platform carries out the first degree analysis to determine whether amino acids are present at all in the rock. Then, if the answer to this question is “yes,” the sample is transferred to the microfabricated, automated lab-on-chip system, for identification and chiral analysis.
How does MOA acquire the sample amino acids from the Martian soil?
The rock to be analyzed is first crushed in a rock crusher. The powdered rock is then transferred to a chamber, which is kept at a constant, low pressure. The sample is heated slowly and different components of the powdered rock sublime at different temperatures. The species in the gas phase then condense onto a cold piece of metal called a “cold finger.”
At what point do the acquired samples get labeled and how does the labeling occur?
The cold finger, onto which the species from the crushed rock condense is previously covered with a layer of the fluorescent dye (fluorescamine is the optimal choice). The dye reacts only with the amine group, thus making only amino acids and other amine-containing molecules “visible” under fluorescence spectroscopy. As a result, at this point, without any further analysis, the presence or absence of amino acids can be established.
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