New Methodology for Accurate Determination of Molecular Co-localization at the Nanoscale.

A new methodology using nanoparticle projectile secondary ion mass spectrometry was developed to identify statistically significant co-localization of tagged proteins versus random aggregations at the nanoscale. The custom instrument was run in the unique event-by-event bombardment detection mode with 1040 keV Au28008+ individual projectiles each probing an area with a diameter of ∼20 nm. In a model experiment, antibodies tagged with fluorine, iodine, and bromine were attached on a silicon wafer in a 1:1:1 ratio. To determine whether the three different antibodies were homogeneously distributed at the nanoscale or if there were fluctuations due to the slightly different physical properties of the tags, a "co-localization factor" was introduced. It is shown for the first time that the differences in the hydrophobicity of the tags induced fluctuations, causing differential attachment of the tags at the nanoscale. When tags with the same physical and chemical properties were used, the analysis of co-localization factors shows that the attachment became random.

Mass Spectrometry of Nanoparticles is Different.

Secondary ion mass spectrometry, SIMS, is a method of choice for the characterization of nanoparticles, NPs. For NPs with large surface-to-volume ratios, heterogeneity is a concern. Assays should thus be on individual nano-objects rather than an ensemble of NPs; however, this may be difficult or impossible. This limitation can be side-stepped by probing a large number of dispersed NPs one-by-one and recording the emission from each NP separately. A large collection of NPs will likely contain subsets of like-NPs. The experimental approach is to disperse the NPs and hit an individual NP with a single massive cluster (e.g., C-60, Au-400). At impact energies of ~1 keV/atom, they generate notable secondary ion (SI) emission. Examination of small NPs (≤20 nm in diameter) shows that the SI emission is size-dependent and impacts are not all equivalent. Accurate identification of the type of impact is key for qualitative assays of core or outer shell composition. For quantitative assays, the concept of effective impacts is introduced. Selection of co-emitted ejecta combined with rejection (anticoincidence) of substrate ions allows refining chemical information within the projectile interaction volume. Last, to maximize the SI signal, small NPs (≤5 nm in diameter) can be examined in the transmission mode where the SI yields are enhanced ~10-fold over those in the (conventional) reflection direction. Future endeavors should focus on schemes acquiring SIs, electrons, and photons concurrently.

SIMS instrumentation and methodology for mapping of co-localized molecules.

We describe an innovative mode for localizing surface molecules. In this methodology, individual C60 impacts at 50 keV are localized using an electron emission microscope, EEM, synchronized with a time-of-flight mass spectrometer for the detection of the concurrently emitted secondary ions. The instrumentation and methodologies for generating ion maps are presented. The performance of the localization scheme depends on the characteristics of the electron emission, those of the EEM and of the software solutions for image analysis. Using 50 keV C60 projectiles, analyte specific maps and maps of co-emitted species have been obtained. The individual impact sites were localized within 1-2 µm. A distinctive feature of recording individual impacts is the ability to identify co-emitted ions which originate from molecules co-located within ~10 nm.

Characteristics of positive and negative secondary ions emitted from [Formula: see text] and [Formula: see text] impacts.

The current limitation for SIMS analyses is insufficient secondary ion yields, due in part to the inefficiency of traditional primary ions. Massive gold clusters are shown to be a route to significant gains in secondary ion yields relative to other commonly used projectiles. At an impact energy of 520 keV, [Formula: see text] is capable of generating an average of greater than ten secondary ions per projectile, with some impact events generating >100 secondary ions. The capability of this projectile for signal enhancement is further displayed through the observation of up to seven deprotonated molecular ions from a single impact on a neat target of the model pentapeptide leu-enkephalin. Positive and negative spectra of leu-enkephalin reveal two distinct emission regimes responsible for the emission of either intact molecular ions with low internal energies or small fragment species. The internal energy distribution for this projectile is measured using a series of benzylpyridinium salts and compared with the small polyatomic projectile [Formula: see text] at 110 keV as well as distributions previously reported for electrospray ionization and fast atom bombardment. These results show that [Formula: see text] offers high secondary ion yields not only for small fragment ions, e.g. CN-, typically observed in SIMS analyses, but also for characteristic molecular ions. For the leu-enkephalin example, the yields for each of these species are greater than unity.

Characterization of individual nano-objects with nanoprojectile-SIMS.

Secondary ion mass spectrometry (SIMS) applied in the event-by-event bombardment/detection mode is uniquely suited for the characterization of individual nano-objects. In this approach, nano-objects are examined one-by-one, allowing for the detection of variations in composition. The validity of the analysis depends upon the ability to physically isolate the nano-objects on a chemically inert support. This requirement can be realized by deposition of the nano-objects on a Nano-Assisted Laser Desorption/Ionization (NALDI™) plate. The featured nanostructured surface provides a support where nano-objects can be isolated if the deposition is performed at a proper concentration. We demonstrate the characterization of individual nano-objects on a NALDI™ plate for two different types of nanometric bacteriophages: Qß and M13. Scanning electron microscope (SEM) images verified that the integrity of the phages is preserved on the NALDI™ substrate. Mass spectrometric data show secondary ions from the phages are identified and resolved from those from the underlying substrate.

Simultaneous detection and localization of secondary ions and electrons from single large cluster impacts.

The use of large cluster primary ions (e.g. C60, Au400) in secondary ion mass spectrometry has become prevalent in recent years due to their enhanced emission of secondary ions, in particular, molecular ions (MW ≤ 1500 Da). The co-emission of electrons with SIs was investigated per projectile impact. It has been found that SI and electrons yields increased with increasing projectile energy and size. The use of the emitted electrons from impacts of C60 for localization has been demonstrated for cholesterol deposited on a copper grid. The instrumentation, methodologies, and results from these experiments are presented.

On the Surface Mapping using Individual Cluster Impacts.

This paper describes the advantages of using single impacts of large cluster projectiles (e.g. C(60) and Au(400)) for surface mapping and characterization. The analysis of co-emitted time-resolved photon spectra, electron distributions and characteristic secondary ions shows that they can be used as surface fingerprints for target composition, morphology and structure. Photon, electron and secondary ion emission increases with the projectile cluster size and energy. The observed, high abundant secondary ion emission makes cluster projectiles good candidates for surface mapping of atomic and fragment ions (e.g., yield >1 per nominal mass) and molecular ions (e.g., few tens of percent in the 500 < m/z < 1500 range).

Photon, Electron and Secondary Ion Emission from Single C(60) keV Impacts.

This paper presents the first observation of coincidental emission of photons, electrons and secondary ions from individual C(60) keV impacts. An increase in photon, electron and secondary ion yields is observed as a function of C(60) projectile energy. The effect of target structure/composition on photon and electron emissions at the nanometer level is shown for a CsI target. The time-resolved photon emission may be characterized by a fast component emission in the UV-Vis range with a short decay time, while the electron and secondary ion emission follow a Poisson distribution.

Examination of nanoparticles via single large cluster impacts.

This study deals with the determination of the relative abundance of the oxide layer in the near-surface volume of aluminum nanoparticles of 50-100 nm in diameter. They are bombarded with a sequence of single projectiles of Au 400(4+) accelerated to 136 keV. The ionized ejecta from each impact are recorded individually which allows identification of ions emitted from a surface volume of approximately 10 nm in diameter and 5-10 nm in depth. The mode of analyzing ejecta individually from each single cluster impact is a means to apply mass spectrometry in nanovolumes.

Simultaneous ejection of two molecular ions from keV gold atomic and polyatomic projectile impacts.

We present the first experimental data on the simultaneous ejection of two molecular ions from the impact of Au(+)(n) (1< or =n< or =4) with energies ranging between 17 and 56 keV. The yields from single phenylalanine (Ph) emission, coemission of two Ph ions, and emission of the Ph dimer were measured. Large increases (1 to 2 orders of magnitude) in coemitted ion yields were observed with increasing projectile energy and complexity. Correlation coefficients were calculated for the coemission of two Ph ions; their behavior suggests differences in emission pathways for bombardment by atomic and polyatomic projectiles.

Auger stimulated ion desorption of negative ions via K-capture radioactive decay.

We report on Auger stimulated ion desorption via Coulomb explosion from surface self-assembled alkylthiol and fluorocarbon molecular layers, triggered by K-capture decay of an imbedded radioactive 55Fe atom. The charge state of the ejecta is determined by charge exchange in binary atomic collisions in bulk and electron tunneling outside the solid, as well as by fragmentation of electronically excited molecules or molecular fragments. We describe the first nonbeam experiments documenting positive and abundant negative ion desorption due solely to core electron excitation after radioactive decay.