Enhancing Ion Signals and Improving Matrix Selection in Time-of-Flight Secondary Ion Mass Spectrometry with Microvolume Expansion Using Large Argon Clusters.

The molecular environment has an important impact on the ionization mechanism in time-of-flight secondary ion mass spectrometry (ToF-SIMS). In complex samples, desorption/ionization, and thus the detection of a molecular signal, can be hampered by molecular entanglement, ionization-suppressive neighbors, or even an unfavorable sample substrate. Here, a method called microvolume expansion is developed to overcome these negative effects. Large argon clusters are able to transfer biomolecules from a target to a collector in vacuum. In this study, argon gas cluster ion beams (Arn+-GCIB with n centered around 3000 or 5000) are used to expand a microvolume from the sample to a collector, which is a material ideally enhancing the ionization yield. The collector is then analyzed using a liquid metal ion gun. The signal amplification factor corresponding to the expansion of phosphatidylcholine (PC) lipid on collectors partially covered with acidic matrices was evaluated as an initial proof of concept. In one experiment, the PC expansion on a pattern of four drop-casted matrix-assisted laser desorption/ionization matrices led to the selection of α-cyano-4-hydroxycinnamic (CHCA) as the optimal candidate for cationic PC detection. The ion signal is increased by at least three orders of magnitude when PC was expanded using 10 keV Ar3000+ and Ar5000+ on a sublimated layer of CHCA. Finally, the expansion of the gray matter of a mouse on different materials (Si, Au-coated Si, CHCA, and polyethylene) was achieved with varying degrees of success, demonstrating the potential of the method to further analyze complex and fragile biological assemblies.

Improvement of biomolecular analysis in thin films using in situ matrix enhanced secondary ion mass spectrometry.

Sensitivity to molecular ions remains a limiting factor for high resolution imaging mass spectrometry of organic and biological materials. Here, we investigate a variant of matrix-enhanced secondary ion mass spectrometry in which the transfer of matrix molecules to the analyte sample is carried out in situ (in situ ME-SIMS). This approach is therefore compatible with both 2D and 3D imaging by SIMS. In this exploratory study, nanoscale matrix layers were sputter-transferred inside our time-of-flight (ToF)-SIMS to a series of thin films of biomolecules (proteins, sugars, lipids) adsorbed on silicon, and the resulting layers were analyzed and depth-profiled. For this purpose, matrix molecules were desorbed from a coated target (obtained by drop-casting or sublimation) using 10 keV Ar3000+ ion beam sputtering, followed by redeposition on a collector carrying the sample to be analyzed. After evaluating the quality of the transfer of six different matrices on bare Si collectors, α-cyano-4-hydroxycinnamic acid (CHCA) was selected for further experiments. The mass spectra and depth profiles obtained from the organic layer prior to and after the sputter-transfer of CHCA were compared, along with those obtained from regular ME-SIMS samples (dried droplets) and, finally, with MALDI data for the same matrix-analyte combinations. Signal amplification factors were calculated by dividing the integrated molecular intensities obtained with or without matrix transfer. While the amplification factors are between 0.5 and 2 for molecules already detected with high intensities in SIMS, such as cholesterol or human angiotensin, other compounds show very large integrated signal amplification, even above two orders of magnitude. This is the case for D-glucose and cardiolipin, for which the molecular ion intensity is low (or very low) under normal SIMS analysis conditions. For such low ionization probability compounds, the beneficial effect of the matrix is unquestionable. Test experiments on mouse brain tissue sections also indicate signal enhancement with the matrix, especially for high mass lipid ions.

Gas Cluster Ion Beam-Assisted Deposition for Fabricating Dry Multilayers Containing Enzyme and Substrate with On-Demand Release.

Gas cluster ion beam (GCIB)-assisted deposition is used to build multilayered protein-based structures. In this process, Ar3000-5000+ clusters bombard and sputter molecules from a reservoir (target) to a collector, an operation that can be sequentially repeated with multiple targets. The process occurs under a vacuum, making it adequate for further sample conservation in the dry state, since many proteins do not have long-term storage stability in the aqueous state. First of all, the stability in time and versatility in terms of molecule selection are demonstrated with the fabrication of peptide multilayers featuring a clear separation. Then, lysozyme and trypsin are used as protein models to show that the activity remaining on the collector after deposition is linearly proportional to the argon ion dose. The energy per atom (E/n) of the Ar clusters is a parameter that was also changed for lysozyme deposition, and its increase negatively affects activity. The intact detection of larger protein molecules by SDS-PAGE gel electrophoresis and a bioassay (trypsin at ≈25 kDa and glucose oxidase (GOx) at ≈80 kDa) is demonstrated. Finally, GOx and horseradish peroxidase, two proteins involved in the same enzymatic cascade, are successively deposited on ß-d-glucose to build an on-demand release material in which the enzymes and the substrate (ß-d-glucose) are combined in a dry trilayer, and the reaction occurs only upon reintroduction in aqueous medium.

Improvement of Lipid Detection in Mouse Brain and Human Uterine Tissue Sections Using In Situ Matrix Enhanced Secondary Ion Mass Spectrometry.

The potential of mass spectrometry imaging, and especially ToF-SIMS 2D and 3D imaging, for submicrometer-scale, label-free molecular localization in biological tissues is undisputable. Nevertheless, sensitivity issues remain, especially when one wants to achieve the best lateral and vertical (nanometer-scale) resolution. In this study, the interest of in situ matrix transfer for tissue analysis with cluster ion beams (Bin+, Arn+) is explored in detail, using a series of six low molecular weight acidic (MALDI) matrices. After estimating the sensitivity enhancements for phosphatidylcholine (PC), an abundant lipid type present in almost any kind of cell membrane, the most promising matrices were softly transferred in situ on mouse brain and human uterine tissue samples using a 10 keV Ar3000+ cluster beam. Signal enhancements up to 1 order of magnitude for intact lipid signals were observed in both tissues under Bi5+ and Ar3000+ bombardment. The main findings of this study lie in the in-depth characterization of uterine tissue samples, the demonstration that the transferred matrices also improve signal efficiency in the negative ion polarity and that they perform as well when using Bin+ and Arn+ primary ions for analysis and imaging.

Gas Cluster Ion Beams as a Versatile Soft-Landing Tool for the Controlled Construction of Thin (Bio)Films.

Surface biofunctionalization with proteins is the key to many biomedical applications. In this study, a solvent-free method for the controlled construction of protein thin films is reported. Using large argon gas cluster ion beams, proteins are sputtered from a target (a pool of pure proteins), and collected on a chosen substrate, being nearly any solid material. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) revealed the presence of intact protein molecules on the collectors. Furthermore, lowering the energy per atom in the cluster projectiles down to 1 eV/atom allowed more than 60% of bradykinin molecules to be transferred intact. This protein deposition method offers a precise control of the film thickness as the transferred protein quantity is proportional to the argon clusters ion dose reached for the transfer. This major feature enables building protein films from (sub)mono- to multilayers, without upper limitation of the thickness. A procedure was developed to measure the film thickness in situ the ToF-SIMS instrument. The versatility and potential of this soft-landing alternative for further applications is demonstrated on the one hand by building a protein thin film at the surface of paper, a substrate hardly compatible with solution-based adsorption methods. On the other hand, the possibility to achieve alternated multilayer buildup is demonstrated with the construction of a bilayer composed of bradykinin and Irganox, with the two layers well separated. These results lay the first stone toward original and complex multilayers that could previously not be considered with solution-based adsorption methods, and this regardless of the substrate nature.