nai

Project Progress

Limulus Amebocyte Lysate (LAL) and Prophenoloxidase system (PPO) — The goal of LAL is to develop very sensitive methods to detect microbial contamination based on the Limulus Amebocyte Lysate (LAL) and Prophenoloxidase system (PPO). These methods have been documented to be able to detect lipopolysaccharide (LPS), glucan and peptidoglycan down to sub-picogram level in pharmaceutical applications. We have adapted them to rock, soil and metallic surfaces. We project application of these methods to known contemporary samples of Earth origin and then to fossilized Earth samples, with the ultimate goal of using these methods to examine Mars meteorites and samples returned from future Mars missions. It is anticipated that methods will continue to be developed that will enable visualization of microbial structures in rock or mineral by “staining” such samples with chromogenic or electron dense substrates for LAL and PPO.

Fluorescent Molecular Probe Derivatization — Our approach is to target specific organic monofunctional groups on a sample and tag them with a fluorescent molecular probe. Once a sample surface has been tagged the spatial distribution of those monofunctional groups can be determined by fluorescence microscopy. This same surface can then subsequently be analyzed by two-step laser microprobe mass spectrometry (mL²MS) to determine the specific molecular species tagged (see below). Since a single fluorphor molecule is typically capable of being consecutively cycled through the fluorescence “excitation-emission” sequence many times, that is it has a low-photobleaching quantum yield, single molecule detection can routinely be achieved by fluorescence microscopy. The spatial resolution limit is determined by the wavelength of the light emission from the fluorphor and is typically sub-micron. Four molecular probes have been determined to be suitable for geological samples, fluorescein-5-isothiocyanate (FITC), o-phthaldialdehyde (OPA), naphthalenedicarboxaldehyde (NDA), and 4-phenylspiro[furan-2(3H),1’-phthalan]3,3’-dione (Fluorescamine). To date we have successfully demonstrated the detection of primary amines and amino acids on individual microtomed thin-sections of carbonaceous meteorites and interplanetary dust particles (Clemett, S. J.; Messenger, S; Thomas-Keprta, K.L.; Wentworth, S.J.; Robinson, G-A; McKay, D.S.; “Spatially Resolved Analysis of Amines Using a Fluorescent Molecular Probe: Molecular Analysis of IDPs,” LPSC XXXIII & Clemett, S.J.; Messenger,, S.; Keller, L.P.; Thomas-Keprta, K.L., McKay, D.S.; “Spatially Resolved Analysis of Amines in Interplanetary Dust Particles Using Fluorescent Molecular Probes,” MetSoc 2002). Because most fluorphors are conjugated and/or aromatic species they are also ideally suited to subsequent analysis by mL²MS (see below).

Microprobe Two-Step Laser Desorption / Laser Ionization Mass Spectrometer (mL²MS) — We are at present testing our new instrument at NASA Johnson Space Center (JSC), and anticipate full operational status by the end of September 2003. The mL²MS is a powerful technique for spatially resolved analysis of sub-attomole concentrations of complex aromatic molecules. By using fluorescent molecular probes to selectively tag particular organic molecules, species ordinarily not amenable to mL²MS analysis (i.e. non-aromatic species such as amino acids) become readily detectable. Moreover, since it is the fluorphor of the molecular probe that undergoes multiphoton resonant ionization, the photoionization cross sections for tagged molecules are approximately constant, allowing for direct quantitation of the results from mL²MS analysis.

Another part of this project is the development and testing of new combinations of fluorescent probes as applied to live or nonfossilized cells and biofilms. When properly used in the appropriate combinations, such probes can provide the following information on single cells, cell colonies, and complex biofilms: (1) physiological status, (2) specific metabolic activities, (3) gene expression, and (4) total cell densities. We have just begun these studies in our new laboratory at JSC to pursue these techniques. We have been able to test this technique and observe microbial activity within desert varnish layers and organic components in Murchison meteorite. The current work is to try this technique on Martian meteorites.

Antibody development techniques and mission applications — Working with NAI at Carnegie Institute, Mars simulated microgravity experiments testing the ELISA and LAL techniques using hopane antibodies were performed on KC-135 flights. Results show microgravity is not an issue in prohibiting antibody reactions using ELISA and LAL. This has applications to spacecraft instrument development using antibody technology for organic and biosignatures.