ToF-SIMS analysis of ultrathin films and their fragmentation patterns.

Organic thin films are of great interest due to their intriguing interfacial and functional properties, especially for device applications such as thin-film transistors and sensors. As their thickness approaches single nanometer thickness, characterization and interpretation of the extracted data become increasingly complex. In this study, plasma polymerization is used to construct ultrathin films that range in thickness from 1 to 20 nm, and time-of-flight secondary ion mass spectrometry coupled with principal component analysis is used to investigate the effects of film thickness on the resulting spectra. We demonstrate that for these cross-linked plasma polymers, at these thicknesses, the observed trends are different from those obtained from thicker films with lower degrees of cross-linking: contributions from ambient carbon contamination start to dominate the mass spectrum; cluster-induced nonlinear enhancement in secondary ion yield is no longer observed; extent of fragmentation is higher due to confinement of the primary ion energy; and the size of the primary ion source also affects fragmentation (e.g., Bi1 versus Bi5). These differences illustrate that care must be taken in choosing the correct primary ion source as well as in interpreting the data.

Quantitative evaluation of perfluorinated alkanethiol molecular order on gold surfaces.

Self-assembled monolayers (SAMs) of perfluoroalkanethiols [CF3(CF2)xCH2CH2SH (x = 3, 5, 7, and 9)] on gold were characterized by x-ray photoelectron spectroscopy (XPS), near edge x-ray absorption fine structure (NEXAFS), and static time-of-flight secondary ion mass spectrometry (ToF-SIMS). Perfluoroalkanethiols of several chain lengths were synthesized using a known hydride reduction method for transforming commercially available perfluoroalkyliodides to corresponding perfluoroalkanethiols. This strategy provides improved product yields compared to other known routes based on hydrolysis from the common thioacetyl perfluoroalkyl intermediate. Angle-dependent XPS analysis revealed that CF3(CF2)xCH2CH2SH (x = 5, 7, and 9; F6, F8, and F10, respectively) SAMs on gold exhibited significant enrichment of the terminal CF3 group at the outer monolayer surface with the sulfur present as a metal-bound thiolate located at the monolayer-gold interface. XPS of the CF3(CF2)3CH2CH2SH (F4) monolayer revealed a thin film with a significant (>50%) amount of hydrocarbon contamination consistent with poorly organized monolayers, while the longest thiol (F10) showed XPS signals attributed to substantial ordering and anisotropy. ToF-SIMS spectra from all four SAMs contained molecular ions representative of the particular perfluorinated thiol used to prepare the monolayer. NEXAFS methods were used to determine degrees of ordering and average tilt for molecules comprising monolayers. The SAMs prepared from the longest (F10) thiols exhibited the highest degree of ordering with the molecular axis nearly perpendicular to the gold surface. The degree of ordering decreased significantly with decreasing length of the perfluorocarbon tail.

XPS and ToF-SIMS Characterization of New Biodegradable Poly(Peptide-Urethane-Urea) Block Copolymers.

New, linear, segmented poly(peptide-urethane-urea) (PPUU) block copolymers are synthesized and their surface compositions are characterized with angle dependent X-ray photoelectron spectroscopy (ADXPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). These new PPUU block copolymers contain three types of segments. The soft segment (SS) is poly(caprolactone diol) (PCL). The hard segment is lysine diisocyanate with a hydrazine chain extender. The oligopeptide segment (OPS) contains three types of amino acids (proline, hydroxyproline, and glycine). Incorporation of the OPS into the polyurethane backbone is done to provide a synthetic polymer material with controllable biodegradation properties. As biodegradation processes normally are initiated at the interface between the biomaterial and the living tissue, it is important to characterize the surface composition of biomaterials. ADXPS and ToF-SIMS results show that the surfaces of all four polymers are enriched with the PCL SS, the most hydrophobic component of the three polymer segments.

Developments and Ongoing Challenges for Analysis of Surface-Bound Proteins.

Proteins at surfaces and interfaces play important roles in the function and performance of materials in applications ranging from diagnostic assays to biomedical devices. To improve the performance of these materials, detailed molecular structure (conformation and orientation) along with the identity and concentrations of the surface-bound proteins on those materials must be determined. This article describes radiolabeling, surface plasmon resonance, quartz crystal microbalance with dissipation, X-ray photoelectron spectroscopy, secondary ion mass spectrometry, sum frequency generation spectroscopy, and computational techniques along with the information each technique provides for characterizing protein films. A multitechnique approach using both experimental and computation methods is required for these investigations. Although it is now possible to gain much insight into the structure of surface-bound proteins, it is still not possible to obtain the same level of structural detail about proteins on surfaces as can be obtained about proteins in crystals and solutions, especially for large, complex proteins. However, recent results have shown it is possible to obtain detailed structural information (e.g., backbone and side chain orientation) about small peptides (5-20 amino sequences) on surfaces. Current studies are extending these investigations to small proteins such as protein G B1 (∼6 kDa). Approaches for furthering the capabilities for characterizing the molecular structure of surface-bound proteins are proposed.

Utilization of chromogenic enzyme substrates for signal amplification in multiplexed detection of biomolecules using surface mass spectrometry.

MicroRNAs (miRNAs) are important post-transcriptional gene regulators and can serve as potential biomarkers for many diseases. Most of the current miRNA detection techniques require purification from biological samples, amplification, labeling, or tagging, which makes quantitative analysis of clinically relevant samples challenging. Here we present a new strategy for the detection of miRNAs with uniformity over a large area based on signal amplification using enzymatic reactions and measurements using time-of-flight secondary ion mass spectrometry (ToF-SIMS), a sensitive surface analysis tool. This technique has high sequence specificity through hybridization with a hairpin DNA probe and allows the identification of single-base mismatches that are difficult to distinguish by conventional mass spectrometry. We successfully detected target miRNAs in biological samples without purification, amplification, or labeling of target molecules. In addition, by adopting a well-known chromogenic enzymatic reaction from the field of biotechnology, we extended the use of enzyme-amplified signal enhancement ToF (EASE-ToF) to protein detection. Our strategy has advantages with respect to scope, quantification, and throughput over the currently available methods, and is amenable to multiplexing based on the outstanding molecular specificity of mass spectrometry (MS). Therefore, our technique not only has the potential for use in clinical diagnosis, but also provides evidence that MS can serve as a useful readout for biosensing to perform multiplexed analysis extending beyond the limitations of existing technology.

Evaluation of surface layer stability of surface-modified polyester biomaterials.

Surface modification of biomaterials is a strategy used to improve cellular and in vivo outcomes. However, most studies do not evaluate the lifetime of the introduced surface layer, which is an important aspect affecting how a biomaterial will interact with a cellular environment both in the short and in the long term. This study evaluated the surface layer stability in vitro in buffer solution of materials produced from poly(lactic-co-glycolic acid) (50:50) and polycaprolactone modified by hydrolysis and/or grafting of hydrophilic polymers using grafting from approaches. The data presented in this study highlight the shortcomings of using model substrates (e.g., spun-coated films) rather than disks, particles, and scaffolds. It also illustrates how similar surface modification strategies in some cases result in very different lifetimes of the surface layer, thus emphasizing the need for these studies as analogies cannot always be drawn.

Versailles Project on Advanced Materials and Standards interlaboratory study on intensity calibration for x-ray photoelectron spectroscopy instruments using low-density polyethylene.

We report the results of a Versailles Project on Advanced Materials and Standards interlaboratory study on the intensity scale calibration of x-ray photoelectron spectrometers using low-density polyethylene (LDPE) as an alternative material to gold, silver, and copper. An improved set of LDPE reference spectra, corrected for different instrument geometries using a quartz-monochromated Al Kα x-ray source, was developed using data provided by participants in this study. Using these new reference spectra, a transmission function was calculated for each dataset that participants provided. When compared to a similar calibration procedure using the NPL reference spectra for gold, the LDPE intensity calibration method achieves an absolute offset of ∼3.0% and a systematic deviation of ±6.5% on average across all participants. For spectra recorded at high pass energies (≥90 eV), values of absolute offset and systematic deviation are ∼5.8% and ±5.7%, respectively, whereas for spectra collected at lower pass energies (<90 eV), values of absolute offset and systematic deviation are ∼4.9% and ±8.8%, respectively; low pass energy spectra perform worse than the global average, in terms of systematic deviations, due to diminished count rates and signal-to-noise ratio. Differences in absolute offset are attributed to the surface roughness of the LDPE induced by sample preparation. We further assess the usability of LDPE as a secondary reference material and comment on its performance in the presence of issues such as variable dark noise, x-ray warm up times, inaccuracy at low count rates, and underlying spectrometer problems. In response to participant feedback and the results of the study, we provide an updated LDPE intensity calibration protocol to address the issues highlighted in the interlaboratory study. We also comment on the lack of implementation of a consistent and traceable intensity calibration method across the community of x-ray photoelectron spectroscopy (XPS) users and, therefore, propose a route to achieving this with the assistance of instrument manufacturers, metrology laboratories, and experts leading to an international standard for XPS intensity scale calibration.

Polymer Surface Analysis: The Leadership and Contributions of David Briggs.

David Briggs was a surface analysis pioneer. Starting in 1970 and continuing throughout his career, Dave used his expertise, vision and ability to quickly master new surface analysis methods and solve important industrial problems. It certainly helped that he was an outstanding fund raiser in both industrial and academic settings, which ensured he always had an impressive array of the latest, most advanced surface analysis instrumentation at his disposal. He insisted on doing surface analysis correctly and through his publications, databases and books he provided the community with the needed guidelines and methods to do so. In the 1970s Dave's research was largely focused on x-ray photoelectron spectroscopy (XPS, also known as electron spectroscopy for chemical analysis (ESCA)) characterization of polymers and catalysts. He added secondary ion mass spectrometry (SIMS) to his instrumentation arsenal in the 1980s and provided many of the key, pioneering publications that described how to use this method to characterize polymer surfaces. He also did some of the first surface analysis imaging experiments in the 1980s. In the 1990s he continued his XPS and SIMS research on polymers and advanced the surface analysis community's ability to properly interpret surface analysis data through data bases and advanced data processing methods. Dave continued to publish polymer and catalysis surface analysis papers in the 2000s, but also expanded his surface analysis studies to several other topics.

Long-term hydrolytic degradation study of polycaprolactone films and fibers grafted with poly(sodium styrene sulfonate): Mechanism study and cell response.

Polycaprolactone (PCL) is a widely used biodegradable polyester for tissue engineering applications when long-term degradation is preferred. In this article, we focused on the analysis of the hydrolytic degradation of virgin and bioactive poly(sodium styrene sulfonate) (pNaSS) functionalized PCL surfaces under simulated physiological conditions (phosphate buffer saline at 25 and 37 °C) for up to 120 weeks with the aim of applying bioactive PCL for ligament tissue engineering. Techniques used to characterize the bulk and surface degradation indicated that PCL was hydrolyzed by a bulk degradation mode with an accelerated degradation-three times increased rate constant-for pNaSS grafted PCL at 37 °C when compared to virgin PCL at 25 °C. The observed degradation mechanism is due to the pNaSS grafting process (oxidation and radical polymerization), which accelerated the degradation until 48 weeks, when a steady state is reached. The PCL surface was altered by pNaSS grafting, introducing hydrophilic sulfonate groups that increase the swelling and smoothing of the surface, which facilitated the degradation. After 48 weeks, pNaSS was largely removed from the surface, and the degradation of virgin and pNaSS grafted surfaces was similar. The cell response of primary fibroblast cells from sheep ligament was consistent with the surface analysis results: a better initial spreading of cells on pNaSS surfaces when compared to virgin surfaces and a tendency to become similar with degradation time. It is worthy to note that during the extended degradation process the surfaces were able to continue inducing better cell spreading and preserve their cell phenotype as shown by collagen gene expressions.

Surface analysis tools for characterizing biological materials.

Surfaces represent a unique state of matter that typically have significantly different compositions and structures from the bulk of a material. Since surfaces are the interface between a material and its environment, they play an important role in how a material interacts with its environment. Thus, it is essential to characterize, in as much detail as possible, the surface structure and composition of a material. However, this can be challenging since the surface region typically is only minute portion of the entire material, requiring specialized techniques to selectively probe the surface region. This tutorial will provide a brief review of several techniques used to characterize the surface and interface regions of biological materials. For each technique we provide a description of the key underlying physics and chemistry principles, the information provided, strengths and weaknesses, the types of samples that can be analyzed, and an example application. Given the surface analysis challenges for biological materials, typically there is never just one technique that can provide a complete surface characterization. Thus, a multi-technique approach to biological surface analysis is always required.

Characterizing protein G B1 orientation and its effect on immunoglobulin G antibody binding using XPS, ToF-SIMS, and quartz crystal microbalance with dissipation monitoring.

Controlling how proteins are immobilized (e.g., controlling their orientation and conformation) is essential for developing and optimizing the performance of in vitro protein-binding devices, such as enzyme-linked immunosorbent assays. Characterizing the identity, orientation, etc., of proteins in complex mixtures of immobilized proteins requires a multitechnique approach. The focus of this work was to control and characterize the orientation of protein G B1, an immunoglobulin G (IgG) antibody-binding domain of protein G, on well-defined surfaces and to measure the effect of protein G B1 orientation on IgG antibody binding. The surface sensitivity of time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to distinguish between different proteins and their orientation on both flat and nanoparticle gold surfaces by monitoring intensity changes of characteristic amino acid mass fragments. Amino acids distributed asymmetrically were used to calculate peak intensity ratios from ToF-SIMS data to determine the orientation of protein G B1 cysteine mutants covalently attached to a maleimide surface. To study the effect of protein orientation on antibody binding, multilayer protein films on flat gold surfaces were formed by binding IgG to the immobilized protein G B1 films. Quartz crystal microbalance with dissipation monitoring and x-ray photoelectron spectroscopy analysis revealed that coverage and orientation affected the antibody-binding process. At high protein G B1 coverage, the cysteine mutant immobilized in an end-on orientation with the C-terminus exposed bound 443 ng/cm2 of whole IgG (H + L) antibodies. In comparison, the high coverage cysteine mutant immobilized in an end-on orientation with the N-terminus exposed did not bind detectable amounts of whole IgG (H + L) antibodies.

A Versatile Catalyst-Free Perfluoroaryl Azide-Aldehyde-Amine Conjugation Reaction.

A tri-component reaction, involving an electrophilically-activated perfluoroaryl azide, an enolizable aldehyde and an amine, reacts readily at room temperature without any catalysts in solvents including aqueous conditions to yield a stable amidine conjugate. The versatility of this reaction is demonstrated in the conjugation of an amino acid without prior protection of the carboxyl group, and in the synthesize antibiotic-nanoparticle conjugates.

Analysis of early cellular responses of anterior cruciate ligament fibroblasts seeded on different molecular weight polycaprolactone films functionalized by a bioactive poly(sodium styrene sulfonate) polymer.

With the growing number of anterior cruciate ligament (ACL) ruptures and the increased interest for regenerative medicine procedures, many studies are now concentrated on developing bioactive and biodegradable synthetic ligaments. For this application, the choice of raw materials with appropriate physicochemical characteristics and long-term degradation features is essential. Polycaprolactone (PCL) has the advantage of slow degradation that depends on its molecular weight. This study evaluates two PCL materials: a technical grade (PC60: 60 kDa) versus a medical grade (PC12: 80 kDa), both before and after functionalization with poly(sodium styrene sulfonate) (pNaSS). After determining the grafting process had little to no effect on the PCL physicochemical properties, sheep ACL fibroblast responses were investigated. The PC12 films induced a significantly lower expression of the tumor necrosis factor alpha inflammatory gene compared to the PC60 films. Both film types induced an overproduction of fibroblast growth factor-2 and transforming growth factor beta compared to the controls on day 5 and demonstrated collagen gene expression profiles similar to the controls on day 7. Upon protein adsorption, pNaSS grafting caused a rapid cell adhesion in the first 30 min and an increased adhesion strength (1.5-fold higher). Moreover, after 7 days, an increase in cell density and actin network development were noted on the grafted films.

Vibrational Sum-Frequency Scattering as a Sensitive Approach to Detect Structural Changes in Collagen Fibers Treated with Surfactants.

Optimizing protocols so that the structure of the collagen fibers in the extracellular matrix remains intact during the decellularization process requires techniques with high structural sensitivity, especially for the surface region of the collagen fibers. Here, we demonstrate that vibrational sum-frequency scattering (SFS) spectroscopy in the protein-specific amide I region provides vibrational spectra and scattering patterns characteristic of protein fiber networks self-assembled in vitro from collagen type I, which are kept in aqueous environments during the analysis. At scattering angles away from the phase-matched direction, the relative strengths of various polarization combinations are highly reproducible, and changes in their ratios can be followed in real time during exposure to sodium dodecyl sulfate surfactant solutions. For the fibers in this work, a scattering angle of about 22° provided specificity for the surface region of the fibers, as it allowed monitoring of immediate structural changes during the surfactant exposure. With further development, we hypothesize that the information from the SFS characterization of collagen fibers may complement information from other techniques with sensitivity to the overall structure, such as second-harmonic generation imaging and infrared spectroscopy, and provide a more complete understanding of fiber molecular structures and interactions during exposure to various environments and conditions.

Structure of von Willebrand factor A1 on polystyrene determined from experimental and calculated sum frequency generation spectra.

The blood-clotting protein von Willebrand factor (vWF) can be activated by small molecules, high shear stress, and interactions with interfaces. It subsequently binds platelet receptor glycoprotein Ibα (GPIbα) at the surface of platelets, thereby playing a crucial role in blood clotting due to platelet activation, which is an important process to consider in the design of cardiovascular implants and biomaterials used in blood-contacting applications. The influence of surfaces on the activation and the molecular-level structure of surface-bound vWF is largely unknown. Recent studies have indicated that when bound to hydrophobic polystyrene (PS), the A1 domain of vWF remains accessible for GPIbα binding. However, the detailed secondary structure and exact orientation of vWF A1 at the PS surface is still unresolved. Here, the authors resolve these features by studying the system with sum-frequency generation (SFG) spectroscopy. The data are consistent with a scenario where vWF A1 maintains a native secondary structure when bound to PS. Comparison of experimental and calculated SFG spectra combined with previously reported time-of-flight secondary ion mass spectrometry data suggests that A1 assumes an orientation with the GPIbα binding domain oriented away from the solid surface and exposed to the solution phase. This structural information will benefit future in vitro experiments with surface-adsorbed A1 domain and may have relevance for the design of novel blood-contacting biomaterials and wound-healing applications.

Surface Analysis: From Single Crystals to Biomaterials.

Surfaces and interfaces play a critical role in material performance in many applications including catalysis, biomaterials, microelectronics, tribology and adhesion. Characterizing the important surfaces and interfaces involved in each application may present different challenges, but the approach to investigating them often is rather similar. Specialized instrumentation is typically used to probe the surface region of a material and often times it is required to develop new instrumentation and data analysis methods to obtain the desired information. It usually best to use multiple experimental techniques, often coupled with theoretical calculations and simulations, to gain a more complete understanding of the surface and interface regions. Careful handling and preparation of the samples is required so the surface is not altered during these processes as well as during analysis. Using model samples with well-defined surface structures and compositions can provide information about fundamental processes as well as help develop the analytical tools and methodology needed to characterize complex surfaces and interfaces. Thus, the expertise and experience a surface analyst acquires in one field can be readily applied to other fields, even when those fields are significantly differently (e.g., biomaterials and microelectronics). This has resulted in surface analysts moving rather easily between different research and application areas. As one example my career path of small molecule chemisorption and reactivity on single crystals to industrial catalysis to biomedical surface science is presented in this manuscript.

Stabilization of dry protein coatings with compatible solutes.

Exposure of protein modified surfaces to air may be necessary in several applications. For example, air contact may be inevitable during the implantation of biomedical devices, for analysis of protein modified surfaces, or for sensor applications. Protein coatings are very sensitive to dehydration and can undergo significant and irreversible alterations of their conformations upon exposure to air. With the use of two compatible solutes from extremophilic bacteria, ectoine and hydroxyectoine, the authors were able to preserve the activity of dried protein monolayers for up to >24 h. The protective effect can be explained by the preferred exclusion model; i.e., the solutes trap a thin water layer around the protein, retaining an aqueous environment and preventing unfolding of the protein. Horseradish peroxidase (HRP) immobilized on compact TiO2 was used as a model system. Structural differences between the compatible solute stabilized and unstabilized protein films, and between different solutes, were analyzed by static time-of-flight secondary ion mass spectrometry (ToF-SIMS). The biological activity difference observed in a colorimetric activity assay was correlated to changes in protein conformation by application of principal component analysis to the static ToF-SIMS data. Additionally, rehydration of the denatured HRP was observed in ToF-SIMS with an exposure of denatured protein coatings to ectoine and hydroxyectoine solutions.

Nonlinear Optical Methods for Characterization of Molecular Structure and Surface Chemistry.

The principles, strengths and limitations of several nonlinear optical (NLO) methods for characterizing biological systems are reviewed. NLO methods encompass a wide range of approaches that can be used for real-time, in-situ characterization of biological systems, typically in a label-free mode. Multiphoton excitation fluorescence (MPEF) is widely used for high-quality imaging based on electronic transitions, but lacks interface specificity. Second harmonic generation (SHG) is a parametric process that has all the virtues of the two-photon version of MPEF, yielding a signal at twice the frequency of the excitation light, which provides interface specificity. Both SHG and MPEF can provide images with high structural contrast, but they typically lack molecular or chemical specificity. Other NLO methods such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) can provide high-sensitivity imaging with chemical information since Raman active vibrations are probed. However, CARS and SRS lack interface and surface specificity. A NLO method that provides both interface/surface specificity as well as molecular specificity is vibrational sum frequency generation (SFG) spectroscopy. Vibration modes that are both Raman and IR active are probed in the SFG process, providing the molecular specificity. SFG, like SHG, is a parametric process, which provides the interface and surface specificity. SFG is typically done in the reflection mode from planar samples. This has yielded rich and detailed information about the molecular structure of biomaterial interfaces and biomolecules interacting with their surfaces. However, 2-D systems have limitations for understanding the interactions of biomolecules and interfaces in the 3-D biological environment. The recent advances made in instrumentation and analysis methods for sum frequency scattering (SFS) now present the opportunity for SFS to be used to directly study biological solutions. By detecting the scattering at angles away from the phase-matched direction even centrosymmetric structures that are isotropic (e.g., spherical nanoparticles functionalized with self-assembled monolayers or biomolecules) can be probed. Often a combination of multiple NLO methods or a combination of a NLO method with other spectroscopic methods is required to obtain a full understanding of the molecular structure and surface chemistry of biomaterials and the biomolecules that interact with them. Using the right combination methods provides a powerful approach for characterizing biological materials.

Grafting of Bioactive Polymers with Various Architectures: A Versatile Tool for Preparing Antibacterial Infection and Biocompatible Surfaces.

The aim of this Research Article is to present three different techniques of poly(sodium styrene sulfonate) (polyNaSS) covalent grafting onto titanium (Ti) surfaces and study the influence of their architecture on biological response. Two of them are "grafting from" techniques requiring an activation step either by thermal or UV irradiation. The third method is a "grafting to" technique involving an anchorage molecule onto which polyNaSS synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization is clicked. The advantage of the "grafting to" technique when compared to the "grafting from" technique is the ability to control the architecture and length of the grafted polymers on the Ti surface and their influence on the biological responses. This investigation compares the effect of the three different grafting processes on the in vitro biological responses of bacteria and osteoblasts. Overall outcomes of this investigation confirmed the significance of the sulfonate functional groups on the biological responses, regardless of the grafting method. In addition, results showed that the architecture and distribution of grafted polyNaSS on Ti surfaces alter the intensity of the bacteria response mediated by fibronectin.