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To combat infections on biomedical devices, antimicrobial coatings have attracted considerable attention, including coatings comprising naturally occurring antimicrobial peptides (AMPs). In this study the aim was to explore performance upon extended challenge by bacteria growing in media above samples. The AMPs LL37, Magainin 2, and Parasin 1 were selected on the basis of well-known membrane disruption activity in solution and were covalently grafted onto a plasma polymer platform, which enables application of this multilayer coating strategy to a wide range of biomaterials. Detailed surface analyses were performed to verify the intended outcomes of the coating sequence. Samples were challenged by incubation in bacterial growth media for 5 and 20 h. Compared with the control plasma polymer surface, all three grafted AMP coatings showed considerable reductions in bacterial colonization even at the high bacterial challenge of initial seeding at 1 &times, 107 CFU, but there were increasing numbers of dead bacteria attached to the surface. All three grafted AMP coatings were found to be non-toxic to primary fibroblasts. These coatings thus could be useful to produce antibacterial surface coatings for biomaterials, though possible consequences arising from the presence of dead bacteria need to be studied further, and compared to non-fouling coatings that avoid attachment of dead bacteria.

While plasma polymerization is used extensively to fabricate functionalized surfaces, the processes leading to plasma polymer growth are not yet completely understood. Thus, reproducing processes in different reactors has remained problematic, which hinders industrial uptake and research progress. Here we examine the crucial role pressure plays in the physical and chemical processes in the plasma phase, in interactions at surfaces in contact with the plasma phase, and how this affects the chemistry of the resulting plasma polymer films using ethanol as the gas precursor. Visual inspection of the plasma reveals a change from intense homogeneous plasma at low pressure to lower intensity bulk plasma at high pressure, but with increased intensity near the walls of the chamber. It is demonstrated that this occurs at the transition from a collision-less to a collisional plasma sheath, which in turn increases ion and energy flux to surfaces at constant RF power. Surface analysis of the resulting plasma polymer films show that increasing the pressure results in increased incorporation of oxygen and lower cross-linking, parameters which are critical to film performance. These results and insights help to explain the considerable differences in plasma polymer properties observed by different research groups using nominally similar processes. Refereed/Peer-reviewed

Poly(ethylene glycol) (PEG) is one of the most extensively studied antifouling coatings due to its ability to reduce protein adsorption and improve biocompatibility. Although the use of PEG for antifouling coatings is well established, the stability and density of PEG layers are often inadequate to provide optimum antifouling properties. To improve on these shortcomings, we employed the stepwise construction of PEG layers onto a silicon surface. Acetylene- terminated alkyl monolayers were attached to nonoxidized crystalline silicon surfaces via a one-step hydrosilylation procedure with 1,8-nonadiyne. The acetylene-terminated surfaces were functionalized via a copper-catalyzed azide − alkyne cycloaddition (CuAAC) reaction of the surface-bound alkynes with an azide to produce an amine terminated layer. The amine terminated layer was then further conjugated with PEG to produce an antifouling surface. The antifouling surface properties were investigated by testing adsorption of human serum albumin (HSA) and lysozyme (Lys) onto PEG layers from phosphate buffer solutions. Detailed characterization of protein fouling was carried out with X-ray photoelectron spectroscopy (XPS) and time-of- flight secondary ion mass spectrometry (ToF-SIMS) combined with principal component analysis (PCA). The results revealed no fouling of albumin onto PEG coatings whereas the smaller protein lysozyme adsorbed to a very low extent. Refereed/Peer-reviewed

This paper investigated the impacts of two deposition times (30 s, 60 s) and discharge powers (20 W, 50 W) on the resultant chemical and physical properties of plasma polymers (pp) formed using heptylamine (HA) or propionaldehyde (PA) monomers. Pinhole-free pp films were formed on all surfaces at deposition rates that varied according to both the time and power employed. XPS and ToF-SIMS analysis showed that power has a more of an effect than time on the chemical properties of the pp, which may be attributed to differences in monomer fragmentation and cross-linking. Mesenchymal stem cell (MSC) adhesion did not vary on the four different HApp surfaces, whereas a negative correlation between cell attachment and increasing plasma energy was observed on PApp surfaces. This suggests that the presence of oxygenated groups, particularly aldehyde and carboxylic groups, may have a dominant impact on MSC adhesion to these functional polymer substrates. Refereed/Peer-reviewed

Time of flight secondary ion mass spectrometry (ToF-SIMS) has been used to determine the extent of surface modification of highly ordered pyrolytic graphite (HOPG) samples that were exposed to radio-frequency methane and hydrogen plasmas. The ToF-SIMS measurements were examined with the multivariate method of principal component analysis (PCA), to maximise the amount of spectral information retained in the analysis. This revealed that the plasma (methane or hydrogen plasma) modified HOPG exhibited greater hydrogen content than the pristine HOPG. The hydrogen content trends observed from the ToF-SIMS studies were also observed in elastic recoil detection analysis measurements. The application of the ToF-SIMS PCA method also showed that small hydrocarbon fragments were sputtered from the hydrogen-plasma-treated sample, characteristic of the formation of a plasma-damaged surface, whereas the methane-plasma-treated surface sputtered larger hydrocarbon fragments, which implies the growth of a polymer-like coating. Scanning tunnelling microscopy measurements of the modified surfaces showed surface features that are attributable to either etching or film growth after exposure to the hydrogen or methane plasma.

Coated layers of biologically active molecules on synthetic biomaterials and biomedical devices can promote a variety of desirable biological reactions by the host body or the biological medium, such as cell and tissue attachment or deterring bacterial biofilm formation. Such coated layers should be immobilised covalently in order to avoid competitive displacement phenomena, and the use of surface-activating plasmas or plasma polymer interlayers with suitable chemical surface groups has proved to be very convenient means of grafting bioactive molecules onto solid materials surfaces. We review selected work on the covalent immobilisation of proteins and peptides onto solid biomaterial surfaces and describe efforts towards plasma methods that allow biomolecules to be covalently captured in a single step. After reviewing a number of approaches, we discuss in more detail the use of plasma polymer interlayers that possess aldehyde or epoxide surface groups; these groups react readily with amine groups on proteins and peptides without undesirable side reactions, and avoid other issues such as crosslinking. We also emphasise the importance of detailed surface analysis to verify that covalent grafting has indeed taken place, and to assess the surface density of grafted molecules. With suitably chosen peptides or proteins, such covalently grafted layers can support the surface attachment of delicate cells, or combat bacterial biofilm formation. Refereed/Peer-reviewed

In the development of bioactive coatings on biomaterials, it is essential to characterize the successful fabrication and the uniformity of intended coatings by sensitive surface analytical techniques, so as to ensure reliable interpretation of observed biointerfacial responses. This can, however, be challenging when small bioactive molecules are grafted onto biomaterials surfaces at sub-and near-monolayer densities. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) provides the required sensitivity, but ion signals from small grafted molecules may still be dominated by fragment ions from the underlying polymer. In such cases, multivariate analysis provides valuable enhancement of spectral data, as illustrated here by examples comprising the surface grafting of bioactive serrulatane molecules, the peptide GRGDSP, the oligonucleotide 15-thymidine, and the antifungal compound Amphotericin B. The authors also show how ToF-SIMS plus principal component analysis can distinguish between covalent grafting and physisorption of the antibiotics caspofungin and micafungin. (C) 2015 American Vacuum Society. Refereed/Peer-reviewed

Not only bacteria but also fungal pathogens, particularly Candida species, can lead to biofilm infections on biomedical devices. By covalent grafting of the antifungal drug caspofungin, which targets the fungal cell wall, onto solid biomaterials, a surface layer can be created that might be able to provide long-term protection against fungal biofilm formation. Plasma polymerization of propionaldehyde (propanal) was used to deposit a thin (~20 nm) interfacial bonding layer bearing aldehyde surface groups that can react with amine groups of caspofungin to form covalent interfacial bonds for immobilization. Surface analyses by x-ray photoelectron spectroscopy and timeof-flight secondary ion mass spectrometry confirmed the intended grafting and uniformity of the coatings, and durability upon extended washing. Testing for fungal cell attachment and ensuing biofilm formation showed that caspofungin retained activity when covalently bound onto surfaces, disrupting colonizing Candida cells. Mammalian cytotoxicity studies using human primary fibroblasts indicated that the caspofungin-grafted surfaces were selective in eliminating fungal cells while allowing attachment and spreading of mammalian cells. These in vitro data suggest promise for use as antifungal coatings, for example, on catheters, and the use of a plasma polymer interlayer enables facile transfer of the coating method onto a wide variety of biomaterials and biomedical devices. Refereed/Peer-reviewed

A block copolymer consisting of a phosphate-containing moiety (poly[2-(methacryloyloxy)ethyl phosphate], PMOEP)and a keto-containing moiety (poly[2-(acetoacetoxy)ethyl methacrylate], PAAEMA) showed good stability after attachment to an APS amine-modified glass slide, as did both of the respective homopolymers. The PAAEMA homopolymer can attach to the APS amine groups via covalent linkages, while the PMOEP homopolymer most likely attaches through electrostatic interactions involving deprotonated phosphate and protonated amine groups. To elucidate the conformation of the block copolymer after attachment, particularly with respect to the PMOEP segment orientation,principal component analysis (PCA) of time-of-flight secondary ion mass spectrometry (ToF-SIMS) spectra of thesurface-attached polymer layers was performed. Comparison with the pure homopolymer spectra and interpretation after PCA indicate that the adsorbed conformation is not random. Rather, the copolymer is adsorbed in a conformation that preferentially exposes the PMOEP block toward the outer surface. We thus conclude that the most likely conformation of PMOEP-b-PAAEMA immobilized onto the APS-modified glass slide is via covalent interfacial linkages involving the PAAEMA block with the result that the surface is enriched in PMOEP tails. This in turn implies that under the conditions applied (dry DMF) the covalent coupling of keto groups to the amine groups of the aminated slide is more efficient than the proton transfer required for the generation of electrostatic attractions. This (partially) preferential orientation of the PMOEP-b-PAAEMA copolymer could have significant implications on interfacial interactions such as those involved in nucleation and the subsequent mineralization sequence of events in hydroxyapatite formation.The present study demonstrates that ToF-SIMS is a powerful tool not only for the investigation of the surface compositionof adsorbed layers, but also for probing the molecular conformation of such adsorbed block copolymers, though careis required in the PCA analysis of multiple spectra. Refereed/Peer-reviewed

A novel plasma functionalization process based on the pulsed plasma polymerization of allyl glycidyl ether is reported for the generation of robust and highly reactive epoxy-functionalized surfaces with well-defined chemical properties. Using a multitechnique approach including X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), infrared spectroscopy (FT-IR), atomic force microscopy (AFM) and ellipsometry, the effect of the plasma deposition parameters on the creation and retention of epoxy surface functionalities was characterized systematically. Under optimal plasma polymerization conditions (duty cycle: 1 ms/20 ms and 1 ms/200 ms), reactive uniform films with a high level of reproducibility were prepared and successfully used to covalently immobilize the model protein lysozyme. Surface derivatization was also carried out with ethanolamine to probe for epoxy groups. The ethanolamine blocked surface resisted nonspecific adsorption of lysozyme. Lysozyme immobilization was also done via microcontact printing. These results show that allyl glycidyl ether plasma polymer layers are an attractive strategy to produce a reactive epoxy functionalized surface on a wide range of substrate materials for biochip and other biotechnology applications. Refereed/Peer-reviewed

A study of polyaniline (PANI), poly-o-toluidine (POT) and poly-o-anisidine (POA) cobalt supported catalysts has been presented. The catalysts were prepared by depositing cobalt species onto the polymers surfaces. Cobalt(II) acetate and N,N′-ethylene-bis(salicylideneimine) cobalt(II) (Co(II)Salen) were used in the syntheses. The catalysts performance was examined in epoxidation of trans-stilbene by molecular oxygen and they turned out to be very efficient under mild conditions. The relationship between the surface exposure of cobalt species and the activity of these polymeric catalysts has been established.

The surface oxidation of metal sulfides in air and aqueous solution is of central importance in mineral separation and environmental control of acid mine drainage. Mechanisms of oxidation, dissolution and surface restructuring have been extensively studied using XPS. High binding energy components in S 2p XPS spectra have been attributed to metal-deficiency, formation of polysulfide S n 2− , elemental sulfur and electronic defect structures (ie Cu(I)/ZnS). The assignment of these components in S 2p XPS spectra has, however, left significant uncertainties particularly in the formation of SS bonding in polysulfide species requiring confirmation from other surface analytical techniques. The use of static ToF-SIMS has provided a new avenue for identification of these species and their development in oxidation of the sulfide surfaces. For the iron sulfides, there is a systematic change in the FeS 2 /FeS fragment ratio from troilite (FeS) through pyrrhotite (Fe 1−x S) to pyrite (FeS 2 ) with ratios varying from 0.59, 1.2 to 32 respectively. Similarly, high ratios for FeS n /FeS are found for pyrite compared with pyrrhotite and troilite mirrored in the S n /S fragment ratios. Changes in surface oxidation, represented in atomic concentrations and S 2p XPS spectra, are seen in the ToF-SIMS signals for S n /SO n ratios in the same iron sulfide sequence. These mass markers, reflecting increased SS bonding, increase in surfaces after oxidation giving further confidence in XPS assignment to polysulfide species. Freshly cleaved galena PbS surfaces reacted in pH8 aqueous solution for increasing periods of time have also shown a systematic increase in S n /S ratios with increasing at.% of oxidised S n 2− species from XPS spectra. Statistical analysis of oxidised galena has shown that the ratios 206 PbO + / 206 Pb + and 208 PbOH + / 208 Pb + directly reflect the degree of oxidation of the surface lead species whilst the O − /S − , S − /total — ion yield and SO 3 − /S − are the best measures for following the oxidation of sulfur species. Results from these ratios suggest that initial air oxidation takes place predominantly on the S sites rather than Pb sites but, in solution at pH9, both sites are oxidised. The ToF-SIMS results appear to directly reflect the surface chemistry of the metal and sulfur species and are not consistent with recombination or fragmentation of secondary neutral or ionic species. The results strongly suggest increasing polymerisation of SS species with increasing oxidation in accord with the XPS assignment to polysulfide of increasing chain length.

Stimuli-responsive materials show considerable promise for applications that require control over biomolecule interactions at solid material interfaces. Graft coatings of poly(N-isopropylacrylamide) (pNIPAM) are of interest for biomedical and biotechnological applications due to their temperature-dependent switching of surface properties between adhesive and nonadhesive states for cells and proteins. The characterization of protein adsorption to these switchable coatings is a formidable task since switching not only influences the affinity for proteins but at the same time induces a significant change in the coating. Here, the highly sensitive analytical technique of time-of-flight-secondary ion mass spectrometry (TOF-SIMS) combined with principal component analysis (PCA) was used for the characterization of protein adsorption onto pNIPAM coatings prepared by free radical polymerization onto surface-bound polymerizable groups. Adsorption of bovine serum albumin and lysozyme onto pNIPAM coatings from phosphate buffered solutions was investigated at temperatures above and below the polymer’s lower critical solution temperature (LCST). Below the LCST, no adsorbed proteins could be detected even with this ultrasensitive method. Whereas above the LCST, adsorbed protein was detected in amounts corresponding at less than the monolayer. PCA loadings plots showed that adventitious contaminants, which might lead to confounding or misleading spectral changes upon protein exposure, were not observed. Refereed/Peer-reviewed