David G. Castner

Imaging time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) has been used to study protein bound to a photolithographically patterned, commercial poly(ethylene glycol) (PEG)‐based polymer film. The effect of different ion sources on the fragmentation pattern from this sample was analyzed with respect to the surface sensitivity of characteristic protein fragments and contrast in the ion images. The method demonstrates that, under similar fluence (below the static limit), Bi ₃ ⁺ provides better surface sensitivity for low mass fragments, and the best image contrast, as compared to Bi ₁ ⁺ and C ₆₀ ⁺ ion sources. Principal component analysis (PCA) was utilized to process depth profiles for this sample and shows that a primary ion fluence of approximately 20 × 10 ¹² ions/cm ² is required to etch through the adsorbed protein layer. Copyright © 2010 John Wiley & Sons, Ltd.

In static SIMS experiments, an analysis dose of 10 ¹² ions/cm ² typically produces optimum results. However, the same dose used in dual beam depth profiling can significantly degrade the signal. This is because during each analysis cycle a high‐energy beam is rastered across the same x ‐ y location on the sample. If a sufficient amount of sample is not removed during each sputter cycle, the subsequent analysis cycle will sample a volume degraded by the previous analysis cycles. The dimensionless parameter, R ′, is used to relate the amount of damage accumulated in the sample to the amount of analysis beam dose used relative to the etching beam. Depth profiles from trehalose films spin‐cast onto silicon wafers acquired using Bi ₁ ⁺ and Bi ₃ ⁺ analysis beams were compared. As R ′ increased, the depth profile and the depth resolution (interface width) both degraded. At R ′ values below 0.04 for both Bi ₁ ⁺ and Bi ₃ ⁺ , the shape of the profile as well as the depth resolution (9 nm) indicated that dual beam analysis can be superior to C ₆₀ single beam depth profiling. Copyright © 2010 John Wiley & Sons, Ltd.

Time‐of‐flight (ToF) SIMS 2D images and molecular depth profiles of human HeLa cells treated with bromodeoxyuridine (BrdU) were acquired in the dual beam mode (Bi ₃ ⁺ analysis beam, C ₆₀ ⁺ etching beam). Several preparation protocols were investigated and were compared to a simple wash‐and‐dry method. The feasibility of using C ₆₀ to clean the samples prior to imaging with Bi was also investigated quantitatively by calibrating full depth profiles of the cells using atomic force microscopy. BrdU was used as a marker for the cell nucleus, facilitating identification and localization of subcellular features during depth profiling. Results show that C ₆₀ can be used to remove the surface contamination and to access different layers within the cells for 2D imaging. For a 1 nA, 10 keV C ₆₀ ⁺ beam incident at 45° and rastered over a 500 × 500 µm ² area, ∼1 nm of biological material was sputtered every second. Our results show that HeLa cells were completely removed after etching with 1.3 × 10 ¹⁵ C ₆₀ ⁺ ions per cm ² , giving an average etching rate of 3.9 nm for every 10 ¹³ C ₆₀ per cm ² at 10 keV and 45° incidence. Copyright © 2010 John Wiley & Sons, Ltd.

The kinetics of adsorption and desorption of two highly asymmetrical model peptides were studied at methyl- and carboxylic acid-terminated alkylthiolate self-assembled monolayer (SAM) surfaces on gold. The model peptides were leucine-lysine (LK), α-helical (LKα14), and β-strand (LKβ15) peptides that have a well-defined secondary structure with the leucines localized on one side and the lysines on the other side. These secondary structures were previously shown to be maintained after adsorption and to control LK peptide orientation on these surfaces. The kinetics of peptide adsorption were analyzed by surface plasmon resonance as a function of peptide solution concentrations at pH 7.4. Peptide desorption was measured by rinsing with a buffer at various times along the adsorption curve. Both peptides had a saturation coverage of approximately 1 ML (monolayer) on the carboxyl SAM. Both peptides exhibited mostly irreversible binding on both surfaces, but the LKα14 peptide showed some limited reversible bindin...

Organophosphonic acid self-assembled monolayers (SAMs) on oxide surfaces have recently seen increased use in electrical and biological sensor applications. The reliability and reproducibility of these sensors require good molecular organization in these SAMs. In this regard, packing, order, and alignment in the SAMs is important, as it influences the electron transport measurements. In this study, we examine the order of hydroxyl- and methyl-terminated phosphonate films deposited onto silicon oxide surfaces by the tethering by aggregation and growth method using complementary, state-of-art surface characterization tools. Near-edge X-ray absorption fine structure (NEXAFS) spectroscopy and in situ sum frequency generation (SFG) spectroscopy are used to study the order of the phosphonate SAMs in vacuum and under aqueous conditions, respectively. X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry results show that these samples form chemically intact monolayer phosphonate films. NEXAFS and SFG spectroscopy showed that molecular order exists in the octadecylphosphonic acid and 11-hydroxyundecylphosphonic acid SAMs. The chain tilt angles in these SAMs were approximately 37° and 45°, respectively.

The ability to orient active proteins on surfaces is a critical aspect of many medical technologies. An important related challenge is characterizing protein orientation in these surface films. This study uses a combination of time-of-flight secondary ion mass spectrometry (ToF-SIMS), sum frequency generation (SFG) vibrational spectroscopy, and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy to characterize the orientation of surface-immobilized Protein G B1, a rigid 6 kDa domain that binds the Fc fragment of IgG. Two Protein G B1 variants with a single cysteine introduced at either end were immobilized via the cysteine thiol onto maleimide-oligo(ethylene glycol)-functionalized gold and bare gold substrates. X-ray photoelectron spectroscopy was used to measure the amount of immobilized protein, and ToF-SIMS was used to measure the amino acid composition of the exposed surface of the protein films and to confirm covalent attachment of protein thiol to the substrate maleimide groups. SFG and NEXAFS were used to characterize the ordering and orientation of peptide or side chain bonds. On both substrates and for both cysteine positions, ToF-SIMS data showed enrichment of mass peaks from amino acids located at the end of the protein opposite to the cysteine surface position as compared with nonspecifically immobilized protein, indicating end-on protein orientations. Orientation on the maleimide substrate was enhanced by increasing pH (7.0−9.5) and salt concentration (0−1.5 M NaCl). SFG spectral peaks characteristic of ordered α-helix and β-sheet elements were observed for both variants but not for cysteine-free wild type protein on the maleimide surface. The phase of the α-helix and β-sheet peaks indicated a predominantly upright orientation for both variants, consistent with an end-on protein binding configuration. Polarization dependence of the NEXAFS signal from the N 1s to π* transition of β-sheet peptide bonds also indicated protein ordering, with an estimated tilt angle of inner β-strands of 40−50° for both variants (one variant more tilted than the other), consistent with SFG results. The combined results demonstrate the power of using complementary techniques to probe protein orientation on surfaces.

The structure, orientation, and formation of amphiphilic α-helix model peptide films on fluorocarbon surfaces has been monitored with sum frequency generation (SFG) vibrational spectroscopy, near-edge x-ray absorption fine structure (NEXAFS) spectroscopy, and x-ray photoelectron spectroscopy (XPS). The α-helix peptide is a 14-mer of hydrophilic lysine and hydrophobic leucine residues with a hydrophobic periodicity of 3.5. This periodicity yields a rigid amphiphilic peptide with leucine and lysine side chains located on opposite sides. XPS composition analysis confirms the formation of a peptide film that covers about 75% of the surface. NEXAFS data are consistent with chemically intact adsorption of the peptides. A weak linear dichroism of the amide π is likely due to the broad distribution of amide bond orientations inherent to the α-helical secondary structure. SFG spectra exhibit strong peaks near 2865 and 2935 cm−1 related to aligned leucine side chains interacting with the hydrophobic surface. Water ...

In dual-beam depth profiling, a high energy analysis beam and a lower energy etching beam are operated in series. Although the fluence of the analysis beam is usually kept well below the static SIMS limit, complete removal of the damage induced by the high energy analysis beam while maintaining a good depth resolution is difficult. In this study, a plasma polymerized tetraglyme film is used as the model organic system and the dimensionless parameter R, (analysis beam fluence)/(total ion fluence), is introduced to quantify the degree of sample damage induced as a function of the analysis beam fluence. For a constant C₆₀⁺ etching beam fluence, increasing the analysis fluence (and consequently increasing the R parameter) increased the amount of damage accumulated in the sample. For Bi_n⁺ (n = 1 and 3) and C₆₀⁺ depth profiling, minimal damage accumulation was observed up to R = 0.03, with a best depth resolution of 8 nm. In general, an increase in the Bi_n⁺ analysis fluence above this value resulted in a decrease in the molecular signals of the steady state region of the depth profile and a degradation of the depth resolution at the polymer/substrate interface.

The structure and orientation of amphiphilic α-helix and β-strand model peptide films on self-assembled monolayers (SAMs) have been studied with sum frequency generation (SFG) vibrational spectroscopy and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. The α-helix peptide is a 14-mer, and the β-strand is a 15-mer of hydrophilic lysine and hydrophobic leucine residues with hydrophobic periodicities of 3.5 and 2, respectively. These periodicities result in the leucine side chains located on one side of the peptides and the lysine side chains on the other side. The SAMs were prepared from the assembly of either carboxylic acid- or methyl-terminated alkyl thiols onto gold surfaces. For SFG studies, the deuterated analog of the methyl SAM was used. SFG vibrational spectra in the C−H region of air-dried peptides films on both SAMs exhibit strong peaks near 2965, 2940, and 2875 cm⁻¹ related to ordered leucine side chains. The orientation of the leucine side chains was determined from the phase of these features relative to the nonresonant gold background. The relative phase for both the α-helix and β-strand peptides showed that the leucine side chains were oriented away from the carboxylic acid SAM surface and oriented toward the methyl SAM surface. Amide I peaks observed near 1656 cm⁻¹ for the α-helix peptide confirm that the secondary structure is preserved on both SAMs. Strong linear dichroism related to the amide π* orbital at 400.8 eV was observed in the nitrogen K-edge NEXAFS spectra for the adsorbed β-strand peptides, suggesting that the peptide backbones are oriented parallel to the SAM surface with the side chains pointing toward or away from the interface. For the α-helix the dichroism of the amide π* is significantly weaker, probably because of the broad distribution of amide bond orientations in the α-helix secondary structure.