nai

Project Progress

The aim of this project is the development of state-of-the-art instrumentation and accompanying protocols for analysis and detection of trace organic matter in terrestrial and astromaterials. Several independent approaches are being developed as outlined below:

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 (µL²MS) 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-emisson” 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 µL²MS (see below).

Microprobe Two-Step Laser Desorption / Laser Ionization Mass Spectrometer (µL²MS) — We are at present assembling a new instrument as NASA Johnson Space Center (JSC), and we anticipate initial operation by the end of September 2002. ²MS 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 L²MS 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 µL²MS analysis.

Time-of-Flight Secondary Ion Mass Spectrometers (TOF-SIMS) — We are currently using Montana State University TOF-SIMS in collaboration with Ricep Avec…..

Aohrpr fized cells and biofilms. This part of the project is managed by John Lisle. 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 are setting up a new laboratory at JSC to pursue these techniques.

A new and very sensitive method to detect microbial traces in rocks is being developed by Norman Wainwright. This technique uses Limulus Amebocyte Lysate and Prophenoloxidase to detect lipopolysaccharide (LPS), glucan, and peptidoglycan down to subpicogram levels. Current efforts involve staining the reacted sample with fixed Limulus anti-LPS factor (LALF) with an antibody that binds to the reacted LALF and a gold-labeled protein and then detecting the stained material with optical and electron microscopes.

Larry Hersman and his team at Los Alamos are continuing to develop sensitive techniques for detecting microbe fossils. Current results show automatic force microscope (AFM) images of fossils as well as X-ray Photoelectron Spectroscopy (XPS) analysis of fossil surfaces.