The ability of mass spectrometry to generate intact biomolecular ions efficiently in the gas phase has led to its widespread application in metabolomics, proteomics, biological imaging, biomarker discovery and clinical assays (namely neonatal screens). Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization have been at the forefront of these developments. However, matrix application complicates the use of MALDI for cellular, tissue, biofluid and microarray analysis and can limit the spatial resolution because of the matrix crystal size (typically more than 10 mum), sensitivity and detection of small compounds (less than 500 Da). Secondary-ion mass spectrometry has extremely high lateral resolution (100 nm) and has found biological applications although the energetic desorption/ionization is a limitation owing to molecular fragmentation. Here we introduce nanostructure-initiator mass spectrometry (NIMS), a tool for spatially defined mass analysis. NIMS uses 'initiator' molecules trapped in nanostructured surfaces or 'clathrates' to release and ionize intact molecules adsorbed on the surface. This surface responds to both ion and laser irradiation. The lateral resolution (ion-NIMS about 150 nm), sensitivity, matrix-free and reduced fragmentation of NIMS allows direct characterization of peptide microarrays, direct mass analysis of single cells, tissue imaging, and direct characterization of blood and urine.

The self-assembly of streptavidin onto biotinylated alkylthiolate monolayers on gold has served as an important model system for protein immobilization at surfaces. Here, we report a detailed study of the surface composition and structure of mixed self-assembled monolayers (SAMs) containing biotinylated and diluent alkylthiolates and their use to specifically immobilize streptavidin. X-ray photoelectron spectroscopy (XPS), angle-resolved XPS (ARXPS), near-edge X-ray absorption fine structure (NEXAFS), and surface plasmon resonance (SPR) have been used to characterize the films produced on gold from a range of binary mixtures of a biotinylated alkylthiol (BAT) and either a C16 methyl-terminated thiol (mercaptohexadecane, MHD) or a C11-oligo(ethylene glycol)-terminated (OEG) thiol in ethanol. The correlation between the solution mole fraction of BAT and its surface mole fraction (χBAT,sur) indicates that it adsorbs ∼4-fold faster than OEG but slightly slower than MHD. ARXPS analysis demonstrates that the biotin terminus of the BAT is exposed at the surface of mixed monolayers with χBAT,sur < 0.5 but is randomly distributed through BAT-rich films. Thus, the OEG diluent not only adds nonfouling properties but induces an improved concentration of biotin at the surface and reduces the exposure of the methylene segments of BAT. NEXAFS characterization demonstrates that pure OEG and mixed BAT/OEG SAMs do not show significant anisotropy in C−C bond orientation, in contrast to MHD and mixed BAT/MHD SAMs, whose aliphatic segments exhibit pseudo-crystalline packing. SPR measurements of streptavidin binding to and competitive dissociation from the different mixed SAMs indicate that streptavidin binds both specifically and nonspecifically to the BAT/MHD SAMs but purely specifically to BAT/OEG SAMs with χBAT,sur < 0.5. For BAT/OEG mixtures with χBAT,sur = 0.1−0.5, specifically bound streptavidin coverages of ∼80% of the C(2,2,2) two-dimensional streptavidin crystalline density (∼280 ng/cm2) can be reproducibly achieved. These composite results clarify the relationship between the specificity of streptavidin recognition and the surface architecture and properties of the mixed SAMs.

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We investigated the composition, properties, and utility of a novel copolymer of P(AAm-co-EG) designed to be an adaptable, durable, and biocompatible surface treatment of metallic, polymeric, and ceramic materials. Solution deposition and photoinitiation reactions were employed to graft a silane layer and then two sequential polymer layers (a discontinuous two stage polymerization) onto oxide surfaces. Different solvents, polymer concentrations, and cross-linker concentrations in the top polymer layer were compared. Contact angle measurements, spectroscopic ellipsometry, and X-ray photoelectron spectroscopy were used to characterize layer wettability, thickness, and chemistry, respectively. A sandwich type network formed between acrylamide and poly(ethylene glycol) when acetone was used as the solvent for both layers. In contrast, an interpenetrating polymer network between acrylamide and poly(ethylene glycol) formed when acetone and methanol were used as the solvents for polymerization of the acrylamide and poly(ethylene glycol) layers, respectively. Interpenetrating polymer network configured samples were tested for protein adsorption and strength of cell attachment. Protein adsorption experiments in 15% fetal bovine serum indicated that significant amounts of protein do not adsorb to the surface of the thin polymer films (∼20 nm). Cell detachment experiments indicated that cells contacting copolymer-modified surfaces were removed by lower shear stresses than cells contacting clean and amine-terminated, (N-(2-aminoethyl)-3-aminopropyl)-trimethoxysilane modified surfaces.

Cell adhesion to biomaterials is a prerequisite for tissue integration with the implant surface. Herein, we show that we can generate a model silica surface that contains a minimal-length arginine-glycine-aspartic acid (RGD) peptide that maintains its biological activity. In the first part of this study, attachment of MC3T3-E1 osteoblast-like cells was investigated on silicon oxide, amine terminated substrates [i.e., 3-aminopropyl triethoxysilane (APTS)], grafted RGD, and physisorbed RGD control. The APTS layer exhibited nanoscale roughness and presented amine functional groups for grafting a minimal RGD tripeptide devoid of any flanking groups or spacers. Contact angle measurements indicated that the hydrophobicity of the APTS surface was significantly lower than that of the surface with grafted RGD (RGD-APTS). Atomic force microscopy showed that surfaces covered with RGD-APTS were smoother (Ra = 0.71 nm) than those covered with APTS alone (Ra = 1.59 nm). Focusing mainly on cell morphology, experiments showed that the RGD-APTS hybrid provided an optimum surface for cell adhesion, spreading, and cytoskeletal organization. Discrete focal adhesion plaques were also observed consistent with successful cell signaling events. In a second set of experiments, smooth, monolayers of APTS (Ra = 0.1 nm) were used to prepare arginine-glycine-aspartic acid-serine (RGDS)-APTS and arginine-glycine-glutamic acid-serine (RGES)-APTS (control) substrates. Focusing mainly on cell function, integrin and gene expression were all enhanced for rate osteosarcoma cells on surfaces containing grafted RGDS. Both sets of studies demonstrated that grafted molecules of RGD(S) enhance both osteoblast-like cell adhesion and function.

Interpenetrating polymer networks (IPNs) of poly(acrylamide-co-ethylene glycol/acrylic acid) (p(AAm-co-EG/AAc) applied to model surfaces prevent protein adsorption and cell adhesion. Subsequently, IPN surfaces functionalized with the RGD cell-binding domain from rat bone sialoprotein (BSP) modulated bone cell adhesion, proliferation, and matrix mineralization. The objective of this study was to utilize the same biomimetic modification strategy to produce functionally similar p(AAm-co-EG/AAc) IPNs on clinically relevant titanium surfaces. Contact angle goniometry and X-ray photoelectron spectroscopy (XPS) data were consistent with the presence of the intended surface modifications. Cellular response was gauged by challenging the surfaces with primary rat calvarial osteoblast (RCO) surfaces in serum-containing media. IPN modified titanium and negative control (RGE-IPN) surfaces inhibit cell adhesion and proliferation, while RGD-modified IPNs on titanium supported osteoblast attachment and spreading. Furthermore, the latter surfaces supported significant mineralization despite exhibiting lower levels of proliferation than positive control surfaces. These results suggest that with the appropriate optimization, this approach may be practical for surface engineering of osseous implants.

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SBA-15 mesoporous silicates with surface functional groups were synthesized by cohydrolysis of tetraethoxyorthosilicate and ethyl-, carboxylate-, and ethylenediaminetriacetic acid-functionalized organosilanes (ETES, CTES, EDATAS, respectively) using the nonionic surfactant poly(ethylene oxide)−poly(propylene oxide)−poly(ethylene oxide) triblock copolymer (PEO20PPO70PEO20, Pluronic P123). X-ray diffraction revealed that silicates synthesized with up to 5% (wt/wt total silica) of added functionalized silane yielded ordered mesoporous materials with P 6 m m hexagonal symmetry. Further increasing the amount of added functionalized silanes to 20% resulted in a significant decrease in the structural ordering of the resulting silicate. Surface areas, pore volumes, and pore diameters were determined from nitrogen gas adsorption/desorption isotherms. Pore size contraction was observed as the wt % of added ETES was increased but not for the silicates formed with added CTES or EDATAS, with the exception of the silicate formed using 20% added EDATAS. Inclusion of 1,3,5-trimethylbenzene (TMB) during synthesis resulted in silicates with larger pore sizes but a loss of structural order. Appreciable adsorption of copper ions from solution was observed only for the EDATAS-functionalized silicates. X-ray photoelectron spectroscopy of Cu2+-bound EDATAS-functionalized silicates revealed an Cu/N ratio of 0.15, smaller than expected for 1:1 stoichiometry of copper ions and etylenediaminetriacetic acid groups. Adsorption isotherms for Cu2+ binding to EDATAS-functionalized silicates were fit to a double-component Langmuir equation. Binding constants, but not capacity, were dependent on the amount of added EDATAS used in the synthesis and were several orders of magnitude smaller than that reported for structurally similar nonimmobilized HEDTA.