PAST RESEARCH

LASER EJECTION OF BIOLOGICAL MOLECULES:

Our research regarding the laser ejection of biological molecules has led to methods to deliver single-stranded DNA up to 2,000 bases in length into the gas phase with no accompanying decomposition.(1,2) This experiment involves coupling an intense laser pulse into a condensed phase system containing the oligonucleotide.(3) In this project we probe the chemical, angular and velocity distributions(4,5) of laser ejected molecules using a number of techniques including, radiography, gel electrophoresis, time-resolved fluorescence and laser postionization, time-of-flight mass spectrometry.(6) One aim is to determine the mechanism by which huge molecules can be transferred into the gas phase intact using laser ejection. All of our evidence to date points toward an electronic mechanism for laser ejection.(4,5,7) Another aim is to channel this research into useful biotechnological experiments. This project has led to two specific biotech applications: DNA sequencing by laser-based mass spectroscopy(8,9) and the analysis of oligonucleotides hybridized to complementary DNA tethered to surfaces. In the DNA sequencing experiment a visible laser is employed to transfer all of the products of a Sanger dideoxy sequencing reaction from a rhodamine 6G matrix into the gas phase, intact. A number of photoionization schemes are under investigation to simultaneously ionize all of the product strands. The ionized strands are then weighed using time-of-flight mass spectroscopy to determine the lengths of the product strands. The information content of this experiment is essentially identical to that obtained using gel electrophoresis. However, in the mass spectroscopic experiment, the strand length determination requires orders of magnitude less time than in the chromatographic experiment. In the second application, concerning laser ejection of oligonucleotides bound to hybridization arrays, we are combining our surface chemical experience with the laser ejection experiment. In essence this experiment involves tethering short oligonucleotides to synthesize arrays of well defined oligonucleotide hybridization sites. These arrays will then be used to demultiplex the products of multiplexed DNA sequencing reactions or multiplexed polymerase chain reaction products. The idea is to spatially separate the various reaction products using hybridization arrays and to analyze those arrays using laser-based mass spectroscopy. This laboratory has recently succeeded in laser ejecting and mass spectroscopically detecting an oligonucleotide hybridized to a surface.

 

THE NATURE OF THE SURFACE-ADSORBATE POTENTIAL ENERGY SURFACE:

In another facet of our investigations we investigate the chemical dynamics of translationally energetic (0.5 to 5eV) atom-surface collisions. Here we have constructed a hyperthermal atomic beam line coupled to an ultrahigh vacuum surface analysis system. The beam line is capable of delivering a high flux of Xe atoms, with up to 5eV kinetic energy, to a well-defined platinum {111} single crystal surface. Many novel experiments can be proposed and performed using this apparatus because the energy deposition in a hyperthermal atom-adsorbate collision proceeds on the 100 femtosecond time scale while the energy dissipation in the bulk lattice is even more rapid. In one scattering experiment we have begun to probe the potential energy surface (PES) of adsorbed molecules. A new method has been developed in this laboratory to determine the well depth (bond energy) of the surface adsorbate PES using collision-induced desorption. We have published a theory(12) for extracting the bond dissociation energy from collision-induced desorption cross section measurements. We applied this theory first to the physisorption system CH4/Ni{111}(12) and have subsequently extended the theory and experimental measurements to the chemisorption systems NH3/Pt{111}(13,14), C2H4/Pt{111}(15) and C2H4/O/Pt{111}. The latter two adsorbate surface systems are of considerable interest for understanding hydrocarbon conversion catalysis. In such systems, the binding energies for are notoriously difficult to obtain using thermal methods because of surface decomposition channels. In one other portion of this experiment we are preparing to create nanowires using collision-induced diffusion. In this experiment the parallel component of kinetic energy in a glancing Xe beam will be employed to promote diffusion of a noble metal overlayer into the step edge of a vicinal metal oxide surface.

REFERENCES:

1.) Levis, R.J., Romano, L.J., "Laser Vaporization of Single-Stranded DNA; A Study of Photoinduced Phosphodiester Bond Cleavage." J. Am. Chem. Soc., 113, 7802, 1991.

2.) Romano, L.J., Levis, R.J., "Nondestructive Laser Vaporization of High Molecular Weight, Single-Stranded DNA." J. Am. Chem Soc., 113, 9665, 1991.

3.) Levis, R.J. "Laser Desorption and Ejection of Biomolecules from the Condensed Phase into the Gas Phase" Annual Review of Physical Chemistry, 1994, 45, 483.

4.) Levis, R.J., Romano, L.J., Rajan, J., Schilke, D., DeWitt, M., "High Speed DNASequencing in the Gas Phase," Proceedings of the SPIE Biomedical OpticsSociety Meeting on Advances in DNA Sequencing, 1992, 1891, 102.

5.) Levis, R.J. and Romano, L.J., "Chemical Products, Angular Distribution and Ion Yield in the Laser Vaporization of Single-Stranded DNA from Thin Films of DNA." submitted to Journal American Chemical Society.

6.) Schilke, D. and Levis, R.J., "A Laser Desorption, Laser Ionization Time-of-Flight Mass Spectrometer for the Interrogation of Fragile Biomolecules," Review of Scientific Instruments, 1994, 65, 1605.

7.) Srinivasan, J., Romano, L. and Levis R.J., "Laser Vaporization and Multiphoton Ionization of an Anthracene-Labeled Nucleotide," J. Phys. Chem., 1995, 99, 13272.

8.) Srinivasan, J., Kool, E.T. and Levis, R.J., "Mass Analysis of Laser-Ejected Linear and Circular DNA: Evidence for a Stable Phosphodiester Backbone During Ejection," submitted to Rapid Commun. Mass Spec.

9.) Srinivasan, J. and Levis, R.J., "Detection of Femtomolar Quantities of DNA Using Laser Ejection/Ionization Time-of-Flight Mass Spectrometry" Analytical Chemistry, submitted

10.) DeWitt, M., Levis, R.J., "Near Infrared Femtosecond Photoionization of Cyclic Aromatic Hydrocarbons," J. Chem. Phys. 1995, 102, 8670.

11.) DeWitt, M., Levis, R.J., "High-Field Ionization of Molecules Using Ultrafast Radiation," Proceedings of the Lausanne Conference on Femtochemistry, in press.

12.) Szulczewski, G., Levis, R.J., "A Theory for Determining Surface-Adsorbate Bond Energies from Desorption Threshold Measurements." J. Chem. Phys. 98(7), 5974, 1993

13.) Szulczewski, G., Levis, R.J., "Determination of a Chemisorption Bond Strength by Direct Measurement of the Threshold Desorption Energy; NH3 on Pt{111}," J. Chem. Phys., 1994, 101, 11070.

14.) Szulczewski, G., Levis, R.J., "Collision-Induced Desorption of Ammonia on Pt{111}: From Direct Measurement of the Threshold Energy to Determination of the Surface-Adsorbate Bond Strength" J. Chem. Phys., in press.

15.) Szulczewski, G., Levis, R.J., "Using Collision-Induced Desorption to Measure the di- Bond Strength of Ethylene Chemisorption on Pt{111}" submitted to J. Am. Chem. Soc.