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Toward a characterization of the molecular origin of the indirect effects in the radiation damage
C. Desfrançois, F. Lecomte, N. Nieuwjaer and B. Manil
The last experimental achievement of the BMS team has been the development of an alternative technique to get biomolecules and their complexes directly under vacuum in conditions as close as the ones encountered in solution. This molecular source is new, does not exist in France and is based on ultra-soft laser desorption from liquid microdroplets under vacuum. The desorbed complexes, initially present in the liquid, can thus be identified and characterized in the gas phase under carefully chosen conditions in a controlled environment, close to their native forms in solution. This source is based on a concept originally developed by B. Brutschy (University of Frankfurt): large biomolecules are put in the gas phase by non-resonant laser desorption on microdroplets. We have initiated the development of such a source and we have obtained some first promising results. In our device, liquid microdroplets (50 micrometers diameter) are generated on demand by a commercial droplet generator (Microdrop) and injected from 300 mbar into a high vacuum chamber (10-6 mbar) through differential pumping stages. Upon irradiation by mid-IR broad band laser pulses (centered on an absorption band of the solvent), analyte ions (and neutrals) are ejected directly under vacuum. The desorption occurs at the entrance of a Paul Trap into which ions are guided by a weak electrostatic field, in order to avoid any fragmentation or activation during collisions with the buffer gas in the trap. Ions are then ejected from the trap, accelerated, focused and detected by microchannel plates in a second vacuum chamber (10-7 mbar). The preliminary results show mass-selected non-covalent complexes, which proves that biomolecular desorption is soft enough to preserve weak molecular interactions. This source then represents an important breakthrough in the field of gas-phase analysis techniques applied to relevant biological systems and will give us the unique opportunity to get insights in the damage processes at the molecular level by the use of gas phase analysis techniques.
By coupling of this experimental set-up with an irradiation platform (see figure), we will irradiate the microdroplets (solvent + biomolecules) by simply charged ions with a kinetic energy of some tenths of keV. This irradiation will create a secondary particles radiolysis cascade (solvated electrons and radicals), only into a superficial layer (first hundreds of nanometers) of the droplet. This corresponds to the physico-chemical stage of the irradiation, which is well-known through the study of water radiolysis. During the next (chemical) stage, the free radicals are going to react with biomolecules in the non-irradiated area. This second stage is far less well characterized for water radiolysis, and almost completely not understood in the case of biomolecules. By mass-analyzing the biomolecular products after irradiation and radiolysis inside the microdroplets, our experimental method will give us the possibility to study, for the first time, these radical chemistry processes linked to indirect effects, being free from any other radiation contribution.
Schematic layout of the experimental apparatus
R. Lozada Garcia, S.D. Leite, F. Lecomte, N. Nieuwjaer, C. Desfrançois and B. Manil (in preparation)
Main financial support: BIORAD (2015-2018, INCa-INSERM)