Peptide Fragmentation and Surface Structural Analysis by Means of ToF-SIMS Using Large Cluster Ion Sources.

Peptide or protein structural analysis is crucial for the evaluation of biochips and biodevices, therefore an analytical technique with the ability to detect and identify protein and peptide species directly from surfaces with high lateral resolution is required. In this report, the efficacy of ToF-SIMS to analyze and identify proteins directly from surfaces is evaluated. Although the physics governing the SIMS bombardment process precludes the ability for researchers to detect intact protein or larger peptides of greater than a few thousand mass unit directly, it is possible to obtain information on the partial structures of peptides or proteins using low energy per atom argon cluster ion beams. Large cluster ion beams, such as Ar clusters and C60 ion beams, produce spectra similar to those generated by tandem MS. The SIMS bombardment process also produces peptide fragment ions not detected by conventional MS/MS techniques. In order to clarify appropriate measurement conditions for peptide structural analysis, peptide fragmentation dependency on the energy of a primary ion beam and ToF-SIMS specific fragment ions are evaluated. It was found that the energy range approximately 6 ≤ E/n ≤ 10 eV/atom is most effective for peptide analysis based on peptide fragments and [M + H] ions. We also observed the cleaving of side chain moieties at extremely low-energy E/n ≤ 4 eV/atom.

Semiempirical Rules To Determine Drug Sensitivity and Ionization Efficiency in Secondary Ion Mass Spectrometry Using a Model Tissue Sample.

There is an increasing need in the pharmaceutical industry to reduce drug failure at late stage and thus reduce the cost of developing a new medicine. Since most drug targets are intracellular, this requires a better understanding of the drug disposition within a cell. Secondary ion mass spectrometry has been identified as a potentially important technique to do this, as it is label-free and allows imaging in 3D with subcellular resolution and recent studies have shown promise for amiodarone. An important analytical parameter is sensitivity, and we measure this in a bovine liver homogenate reference sample for 20 drugs representing important class types relevant to the pharmaceutical industry. We also measure the sensitivity for pure drug and show, for the first time, that the secondary ion mass spectrometry (SIMS) positive ionization efficiency for small molecules is a simple power-law relationship to the log P value. This discovery will be important for advancing the understanding of the SIMS ionization process in small molecules that has, until now, been elusive. This simple relationship is found to hold true for drug doped in the bovine liver homogenate reference sample, except for fluticasone, nicardipine, and sorafenib which suffer from severe matrix suppression. This relationship provides a simple semiempirical method to determine drug sensitivity for positive secondary ions. Furthermore, we show, on chosen models, how the use of different solvents during sample preparation can affect the ionization of analytes.

VAMAS interlaboratory study for desorption electrospray ionization mass spectrometry (DESI MS) intensity repeatability and constancy.

A VAMAS (Versailles Project on Advanced Materials and Standards) interlaboratory study for desorption electrospray ionization mass spectrometry (DESI MS) measurements has been conducted with the involvement of 20 laboratories from 10 countries. Participants were provided with an analytical protocol and two reference samples: a thin layer of Rhodamine B and double-sided adhesive tape, each on separate glass slides. The studies comprised acquisition of positive ion mass spectra in predetermined m/z ranges. No sample preparation was required. Results for Rhodamine B show that very consistent craters may be generated. However, inadequacies of the spray and sample stage designs often lead to variable crater shapes. The average repeatability for Rhodamine B is 50%. Yet, repeatabilities better than 20% can be achieved. Rhodamine B proved to be an excellent reference sample to check the sample erosion crater, the sample stage movement and memory effects. Adhesive tape samples show that their average absolute intensity repeatability is 30% and the relative repeatability is 9%. The constancy of these spectra from relative intensities gives day-to-day average relative repeatabilities of 31%, three times worse than the short-term repeatability. Significant differences in the spectra from different laboratories arise from the different adventitious adducts observed or from contaminants that may cause the higher day-to-day variations. It is thought that this may be overcome by allowing some 20 ppb of sodium to be always present in the solvent, to be the dominating adduct. Repeatabilities better than 5% may be achieved with adequate control.

Argon cluster ion beams for organic depth profiling: results from a VAMAS interlaboratory study.

The depth profiling of organic materials with argon cluster ion sputtering has recently become widely available with several manufacturers of surface analytical instrumentation producing sources suitable for surface analysis. In this work, we assess the performance of argon cluster sources in an interlaboratory study under the auspices of VAMAS (Versailles Project on Advanced Materials and Standards). The results are compared to a previous study that focused on C(60)(q+) cluster sources using similar reference materials. Four laboratories participated using time-of-flight secondary-ion mass spectrometry for analysis, three of them using argon cluster sputtering sources and one using a C(60)(+) cluster source. The samples used for the study were organic multilayer reference materials consisting of a ∼400-nm-thick Irganox 1010 matrix with ∼1 nm marker layers of Irganox 3114 at depths of ∼50, 100, 200, and 300 nm. In accordance with a previous report, argon cluster sputtering is shown to provide effectively constant sputtering yields through these reference materials. The work additionally demonstrates that molecular secondary ions may be used to monitor the depth profile and depth resolutions approaching a full width at half maximum (fwhm) of 5 nm can be achieved. The participants employed energies of 2.5 and 5 keV for the argon clusters, and both the sputtering yields and depth resolutions are similar to those extrapolated from C(60)(+) cluster sputtering data. In contrast to C(60)(+) cluster sputtering, however, a negligible variation in sputtering yield with depth was observed and the repeatability of the sputtering yields obtained by two participants was better than 1%. We observe that, with argon cluster sputtering, the position of the marker layers may change by up to 3 nm, depending on which secondary ion is used to monitor the material in these layers, which is an effect not previously visible with C(60)(+) cluster sputtering. We also note that electron irradiation, used for charge compensation, can induce molecular damage to areas of the reference samples well beyond the analyzed region that significantly affects molecular secondary-ion intensities in the initial stages of a depth profile in these materials.

Identification and separation of protein, contaminant and substrate peaks using gentle-secondary ion mass spectrometry and the g-ogram.

RATIONALE: Secondary ion mass spectrometry (SIMS) is an important technique for the characterization of proteins at surfaces. However, interpretation of the mass spectra is complicated owing to confusion with peaks from contaminants and the substrate which is further compounded by complex fragmentation mechanisms. We test a new development of the G-SIMS method called the g-ogram to separate out spectral components without a priori information about which peaks to include in the analysis and which peaks relate to each component. METHODS: The effectiveness of the g-ogram method is investigated using a model system of lysozyme adsorbed onto a silicon wafer and indium tin oxide substrates. In the method, two SIMS spectra are acquired using Bi(+) and Mn(+) primary ions which create lower and higher fragmentation in the spectra, respectively. The g-ogram separates out components using a separation parameter that is related to the fragmentation energy. RESULTS: The g-ogram separates the spectrum of lysozyme adsorbed onto a silicon wafer into three components: (i) the substrate and PDMS contamination; (ii) a second, but unexpected, contaminant; and (iii) peaks from the protein amino acids. Similar results are achieved for the indium tin oxide substrate. In addition, evidence of fragments from plural amino acids with two candidate peaks at 140.12 Da and 185.08 Da is observed. CONCLUSIONS: The g-ogram method effectively separates out mass peaks relating to the substrate, contamination and protein without any a priori information or subjective decisions about which peaks to include in the analysis (so called 'peak picking'). This is a great help to analysts. We find two possible peaks from plural amino acids but no evidence of pluralities is found for peaks above 240 Da that are generated from when using Bi or Mn primary ions.

Modelling of surface nanoparticle inclusions for nanomechanical measurements by an AFM or nanoindenter: spatial issues.

Finite element analysis (FEA) is used to model nanoindentation by a rigid, spherically shaped indenter, axially indenting an elastic two phase polymer system comprised of a cylindrical nanoparticle of compliant polymer set in a semi-infinite matrix of stiffer polymer. The cylindrical nanoparticle is normal to the sample surface. An axisymmetric finite element model is used to determine the reduced modulus measured as a function of the indentation depth for various nanoparticle radii and extensions below the surface. We show how the previous simple analytical equations may be extended to describe these situations with accuracy. This gives excellent agreement with the FEA and provides a clear guide to the maximum indentation depth as a function of both the nanoparticle radius and its thickness consistent with a choice of either computation from the analytical equations or direct measurement with a maximum of 10% error in the measured reduced modulus.

Nanoindentation measurement of Young's modulus for compliant layers on stiffer substrates including the effect of Poisson's ratios.

Finite element analysis (FEA) is used to investigate the effect of the Poisson's ratios of both the overlayer and the substrate on the nanoindentation of an elastic two-phase system where the elastic overlayer is more compliant than the underlying elastic substrate. A rigid spherical indenter is used as a probe. It is found that nanoindentation results may be expressed analytically using a simple extension of the previously described equation (Clifford and Seah 2006 Nanotechnology 17 5283). This simple function describes the reduced modulus value measured using Oliver and Pharr's method (1992 J. Mater. Res. 7 1564) for any modulus values or Poisson's ratio values of the overlayer and substrate, overlayer thickness or indenter tip radius. This equation and the FEA behind it are tested using experimental published data for the nanoindentation of a silicon dioxide layer on silicon.

Chemical Imaging of Buried Interfaces in Organic-Inorganic Devices Using Focused Ion Beam-Time-of-Flight-Secondary-Ion Mass Spectrometry.

Organic-inorganic hybrid materials enable the design and fabrication of new materials with enhanced properties. The interface between the organic and inorganic materials is often critical to the device's performance; therefore, chemical characterization is of significant interest. Because the interfaces are often buried, milling by focused ion beams (FIBs) to expose the interface is becoming increasingly popular. Chemical imaging can subsequently be obtained using secondary-ion mass spectrometry (SIMS). However, the FIB milling process damages the organic material. In this study, we make an organic-inorganic test structure to develop a detailed understanding of the processes involved in FIB milling and SIMS imaging. We provide an analysis methodology that involves a "clean-up" process using sputtering with an argon gas cluster ion source to remove the FIB-induced damage. The methodology is evaluated for two additive manufactured devices, an encapsulated strain sensor containing silver tracks embedded in a polymeric material and a copper track on a flexible polymeric substrate created using a novel nanoparticle sintering technique.

Quantitative molecular depth profiling of organic delta-layers by C60 ion sputtering and SIMS.

Alternating layers of two different organic materials, Irganox1010 and Irganox3114, have been created using vapor deposition. The layers of Irganox3114 were very thin ( approximately 2.5 nm) in comparison to the layers of Irganox1010 ( approximately 55 or approximately 90 nm) to create an organic equivalent of the inorganic 'delta-layers' commonly employed as reference materials in dynamic secondary ion mass spectrometry. Both materials have identical sputtering yields, and we show that organic delta layers may be used to determine some of the important metrological parameters for cluster ion beam depth profiling. We demonstrate, using a C(60) ion source, that the sputtering yield, S, diminishes with ion dose and that the depth resolution also degrades. By comparison with atomic force microscopy data for films of pure Irganox1010, we show that the degradation in depth resolution is caused by the development of topography. Secondary ion intensities are a well-behaved function of sputtering yield and may be employed to obtain useful analytical information. Fragments characteristic of highly damaged material have intensity proportional to S, and those fragments with minimal molecular rearrangment exhibit intensities proportional to S(2). We demonstrate quantitative analysis of the amount of substance in buried layers of a few nanometer thickness with an accuracy of approximately 10%. Organic delta layers are valuable reference materials for comparing the capabilities of different cluster ion sources and experimental arrangements for the depth profiling of organic materials.

Systematic Temperature Effects in the Argon Cluster Ion Sputter Depth Profiling of Organic Materials Using Secondary Ion Mass Spectrometry.

A study is presented of the effects of sample temperature on the sputter depth profiling of two organic materials, NPB (N,N'-Di(1-naphthyl)-N,N'-diphenyl-(1,1'-biphenyl)-4,4'-diamine) and Irganox 1010, using a 5 keV Ar2000 (+) cluster ion beam and analysis by secondary ion mass spectrometry. It is shown that at low temperatures, the yields increase slowly with temperature in accordance with the Universal Sputtering Yield equation where the energy term is now modified by Trouton's rule. This occurs up to a transition temperature, T T, which is, in turn, approximately 0.8T M, where T M is the sample melting temperature in Kelvin. For NPB and Irganox 1010, these transition temperatures are close to 15 °C and 0 °C, respectively. Above this temperature, the rate of increase of the sputtering yield rises by an order of magnitude. During sputtering, the depth resolution also changes with temperature with a very small change occurring below T T. At higher temperatures, the depth resolution improves but then rapidly degrades, possibly as a result first of local crater surface diffusion and then of bulk inter-diffusion. The secondary ion spectra also change with temperature with the intensities of the molecular entities increasing least. This agrees with a model in which the molecular entities arise near the crater rim. It is recommended that for consistent results, measurements for organic materials are always made at temperatures significantly below T T or 0.8 T M, and this is generally below room temperature. Graphical Abstract ᅟ.

Electron flood gun damage effects in 3D secondary ion mass spectrometry imaging of organics.

Electron flood guns used for charge compensation in secondary ion mass spectrometry (SIMS) cause chemical degradation. In this study, the effect of electron flood gun damage on argon cluster depth profiling is evaluated for poly(vinylcarbazole), 1,4-bis((1-naphthylphenyl)amino)biphenyl and Irganox 3114. Thin films of these three materials are irradiated with a range of doses from a focused beam of 20 eV electrons used for charge neutralization. SIMS chemical images of the irradiated surfaces show an ellipsoidal damaged area, approximately 3 mm in length, created by the electron beam. In depth profiles obtained with 5 keV Ar(2000)(+) sputtering from the vicinity of the damaged area, the characteristic ion signal intensity rises from a low level to a steady state. For the damaged thin films, the ion dose required to sputter through the thin film to the substrate is higher than for undamaged areas. It is shown that a damaged layer is formed and this has a sputtering yield that is reduced by up to an order of magnitude and that the thickness of the damaged layer, which increases with the electron dose, can be as much as 20 nm for Irganox 3114. The study emphasizes the importance of minimizing the neutralizing electron dose prior to the analysis.

Depth resolution, angle dependence, and the sputtering yield of Irganox 1010 by coronene primary ions.

A study is reported of the depth resolution and angle dependence of sputtering yields using the reference organic material, Irganox 1010, for a new coronene(+) depth profiling ion source at 8 and 16 keV beam energies. This source provides excellent depth profiles as shown by 8.5 nm marker layers of Irganox 3114. Damage occurs but may be ignored for angles of incidence above 70° from the surface normal, as shown by X-ray photoelectron spectroscopy (XPS) of the C 1s peak structure. Above 70°, XPS profiles of excellent depth resolution are obtained. The depth resolution, after removal of the thickness of the delta layers, shows a basic contribution of 5.7 nm together with a contribution of 0.043 times the depth sputtered. This is lower than generally reported for cluster sources. The coronene(+) source is thus found to be a useful and practical source for depth profiling organic materials. The angle dependencies of both the undamaged and damaged materials are described by a simple equation. The sputtering yields for the undamaged material are described by a universal equation and are consistent with those obtained for C60(+) sputtering. Comparison with the sputtering yields using an argon gas cluster ion source shows great similarities, but the yields for both the coronene(+) and C60(+) primary ion sources are slightly lower.

Nanomechanical measurements of hair as an example of micro-fibre analysis using atomic force microscopy nanoindentation.

The characterisation of nanoscale surface properties of textile and hair fibres is key to developing new effective laundry and hair care products. Here, we develop nanomechanical methods to characterise fibres using an atomic force microscope (AFM) to give their nanoscale modulus. Good mounting methods for the fibre that are chemically inert, clean and give strong mechanical coupling to a substrate are important and here we detail two methods to do this. We show, for elastic nanoindentation measurements, the situation when the tip radius significantly affects the result via a function of the ratio of the radii of the tip and fibre and indicate the importance of using an AFM for such work. A valid method to measure the nanoscale modulus of fibres using AFM is thus detailed and exampled on hair to show that bleaching changes the nanoscale reduced modulus at the outer surface.

Topography and field effects in secondary ion mass spectrometry--part I: conducting samples.

Quantitative chemical characterization of surfaces with topography by secondary ion mass spectrometry (SIMS) remains a significant challenge due to the lack of systematic and validated measurement methods. In this study, we combine an experimental approach using a simple model system with computer simulation using SIMION, to understand and quantify the key factors that give rise to unwanted topographic artefacts in SIMS images of conducting samples with microscale topography. Experimental data are acquired for gold wires (diameters 33 to 125 µm) mounted onto silicon wafers. Significant loss of ion intensities and shadowing arise from the distortion of the extraction field, and the chemical analysis over the whole of the sample surface is difficult. For large primary ion incidence angles of ≥55° to the surface normal, a fraction of the primary ions are scattered from the target and impact the substrate, emitting secondary ions that may be mistaken as originating from the wire. For conducting samples, topographic field effects may be reduced by the use of a smaller extraction voltage and an extraction delay. The effects of an extraction delay on ion intensities, mass resolution and time-of-flight are studied, and its application is demonstrated on an anisotropically etched silicon sample. The use of a simple sample holder with a V-shaped groove to reduce topographic field effects for wires is also presented. Using these results, we provide clear guidance to analysts for the diagnosis and identification of topography effects in SIMS, and present key recommendations to minimize them in practical analysis.

Relationships between cluster secondary ion mass intensities generated by different cluster primary ions.

Measurements are described to evaluate the constitution of secondary ion mass spectra for both monatomic and cluster primary ions. Previous work shows that spectra for different primary ions may be accurately described as the product of three material-dependent component spectra, two being raised to increasing powers as the cluster size increases. That work was for an organic material and, here, this is extended to (SiO(2))(t)OH(-) clusters from silicon oxide sputtered by 25 keV Bi(n)(+) cluster primary ions for n = 1, 3, and 5 and 1 < or = t < or = 15. These results are described to a standard deviation of 2.4% over 6 decades of intensity by the product of a constant with a spectrum, H(SiOH)*, and a power law spectrum in t. This evaluation is extended, using published data for Si(t)(+) sputtered from Si by 9 and 18 keV Au(-) and Au(3)(-), with confirmation that the spectra are closely described by the product of a constant with a spectrum, H(Si)*, and a simple spectrum that is an exponential dependence on t, both being raised to appropriate powers. This is confirmed with further published data for 6, 9, 12, and 18 keV Al(-) and Al(2)(-) primary cluster ions. In all cases, the major effect of intensity is then related to the deposited energy of the primary ion at the surface. The constitution of SIMS spectra, for monatomic and cluster primary ion sources, is shown, in all cases, to be consistent with the product of a constant with two component spectra raised to given powers.

G-SIMS: relative effectiveness of different monatomic primary ion source combinations.

An analysis is made of the characteristics of monatomic primary ion sources to generate G-SIMS (gentle SIMS) spectra. In previous studies, this is resolved into the parameter beta that describes the relative intensities of ions in the series C(n)H(n+2-i) as i changes. For this, data from polystyrene are most extensive. It is found that the experimental beta values, which relate to the emitted secondary ion fragment surface plasma temperatures, are accurately described by an empirical fit involving the ratio of the sputtering yield and the mass of the primary ion. This description covers data for Ar(+), Bi(+), Cs(+), Ga(+), Mn(+) and Xe(+) monatomic primary ions with energies in the range 4 to 25 keV, placing them in a coherent framework, and permits the performance of any other monatomic primary ion to be predicted. This shows that, of all monatomic primary ions, Bi will yield the highest beta values and Mn the lowest. Since the G-SIMS spectra are ratios, a ratio involving spectra using these primary ions gives the maximum signal quality possible and these are therefore recommended for use. The previous choice of these ions for a combined G-SIMS source, based on practical considerations, is thus shown to be optimum.

Cluster ion beam profiling of organics by secondary ion mass spectrometry--does sodium affect the molecular ion intensity at interfaces?

The use of cluster ion beam sputtering for depth profiling organic materials is of growing technological importance and is a very active area of research. At the 44th IUVSTA Workshop on "Sputtering and Ion Emission by Cluster Ion Beams", recent results were presented of a cluster ion beam depth profile of a thin organic molecular layer on a silicon wafer substrate. Those data showed that the intensity of molecular secondary ions is observed to increase at the interface and this was explained in terms of the higher stopping power in the substrate and a consequently higher sputtering yield and even higher secondary ion molecular sputtering yield. An alternative hypothesis was postulated in the workshop discussion which may be paraphrased as: "under primary ion bombardment of an organic layer, mobile ions such as sodium may migrate to the interface with the inorganic substrate and this enhancement of the sodium concentration increases the ionisation probability, so increasing the molecular ion yield observed at the interface". It is important to understand if measurement artefacts occur at interfaces for quantification as these are of great technological relevance - for example, the concentration of drug in a drug delivery system. Here, we evaluate the above hypothesis using a sample that exhibits regions of high and low sodium concentration at both the organic surface and the interface with the silicon wafer substrate. There is no evidence to support the hypothesis that the probability of molecular secondary ion ionisation is related to the sodium concentration at these levels.

Imaging G-SIMS: a novel bismuth-manganese source emitter.

G-SIMS (gentle-SIMS) is a powerful method that considerably simplifies complex static secondary ion mass spectrometry (SSIMS) analysis of organics at surfaces. G-SIMS uses two primary ion beams that generate high and low fragmentation conditions at the surface. This allows an extrapolation to equivalent experimental conditions with very low fragmentation. Consequently, the spectra are less complex, contain more structural information and are simpler to interpret. In general, G-SIMS spectra more closely resemble electron ionisation mass spectra than SSIMS spectra. A barrier for the wider uptake of G-SIMS is the requirement for two ion beams producing suitably different fragmentation conditions and the need for their spatial registration (spatial alignment) at the surface, which is especially important for heterogeneous samples. The most popular source is the liquid metal ion source (LMIS), which is now sold with almost every new time-of-flight (TOF)-SIMS instrument. Here, we have developed a novel bismuth-manganese emitter (the 'G-tip') for the popular LMISs. This simplifies the alignment and gives excellent G-SIMS imaging and spectroscopy without significantly compromising the bismuth cluster ion beam performance.