Helmut Thissen

Stimuli-responsive interfaces and systems for the control of protein-surface and cell-surface interactions

Real-time control over and reversibility of biomolecule-surface interactions at interfaces is an increasingly important goal for a range of scientific fields and applications. The field of stimuli-responsive, smart or switchable systems has generated much research interest due to its potential to attain unprecedented levels of control over biomolecule adsorption processes and interactions at engineered interfaces, including the control over reversibility of adsorption. Advances in this field are particularly relevant to applications in the areas of biosensing, chromatography, drug delivery and regenerative medicine. The control over biomolecule adsorption and desorption processes at interfaces is often used to control subsequent events such as cell-surface interactions. Considerable research interest has been directed at systems that can be reversibly switched between interacting and non-interacting states and used thus for switching, on and off, bio-interfacial interactions such as protein adsorption. Such switchable coatings often incorporate features such as temporal resolution, spatial resolution and reversibility. Here we review recent literature on switchable coatings that employ stimuli such as light, temperature, electric potential, pH and ionic strength to control protein adsorption/desorption and cell attachment/detachment en route to the development of next-generation smart bio-interfaces.

Ultrasensitive probing of the protein resistance of PEG surfaces by secondary ion mass spectrometry

The highly sensitive surface analytical techniques X-ray photoelectron spectroscopy (XPS) and time-of-flight static secondary ion mass spectrometry (ToF-SIMS) were used to test the resistance of poly(ethylene glycol) (PEG) coatings towards adsorption of lysozyme (LYS) and fibronectin (FN). PEG coatings were prepared by grafting methoxy-terminated aldehyde-PEG (MW 5000 Da) onto two amino-functionalised surfaces with different amine group densities, generated by radio frequency glow discharge polymerisation of n-heptylamine and allylamine. Grafting was performed at the lower critical solution temperature to maximise the graft density of the PEG chains. XPS showed that the grafted density of PEG chains was slightly higher on the allylamine surface. XPS detected no adsorption of either protein on either PEG coating. ToF-SIMS analysis, on the other hand, found, in the positive ion spectra, minute but statistically significant signals assignable to amino acid fragment ions from both proteins adsorbed to the lower density PEG coating and from LYS but not FN on the higher density PEG coating. Negative ion spectra contained relatively more intense protein fragment ion signals for the lower density PEG coating but no changes assignable to adsorbed proteins on the higher density PEG coating. These results demonstrate the importance of utilising highly sensitive techniques to study protein adsorption on surfaces intended to be protein resistant, and that both positive and negative ion ToF-SIMS spectra should be acquired to probe for possible very low levels of protein adsorption.

Emerging rules for effective antimicrobial coatings

In order to colonize abiotic surfaces, bacteria and fungi undergo a profound change in their biology to form biofilms: communities of microbes embedded into a matrix of secreted macromolecules. Despite strict hygiene standards, biofilm-related infections associated with implantable devices remain a common complication in the clinic. Here, the application of highly dosed antibiotics is problematic in that the biofilm (i) provides a protective environment for microbes to evade antibiotics and/or (ii) can provide selective pressure for the evolution of antibiotic-resistant microbes. However, recent research suggests that effective prevention of biofilm formation may be achieved by multifunctional surface coatings that provide both non-adhesive and antimicrobial properties imparted by antimicrobial peptides. Such coatings are the subject of this review.

The influence of nanostructured materials on biointerfacial interactions

Control over biointerfacial interactions in vitro and in vivo is the key to many biomedical applications: from cell culture and diagnostic tools to drug delivery, biomaterials and regenerative medicine. The increasing use of nanostructured materials is placing a greater demand on improving our understanding of how these new materials influence biointerfacial interactions, including protein adsorption and subsequent cellular responses. A range of nanoscale material properties influence these interactions, and material toxicity. The ability to manipulate both material nanochemistry and nanotopography remains challenging in its own right, however, a more in-depth knowledge of the subsequent biological responses to these new materials must occur simultaneously if they are ever to be affective in the clinic. We highlight some of the key technologies used for fabrication of nanostructured materials, examine how nanostructured materials influence the behavior of proteins and cells at surfaces and provide details of important analytical techniques used in this context.

Patterned and switchable surfaces for biomolecular manipulation

The interactions of biomolecules and cells with solid interfaces play a pivotal role in a range of biomedical applications and have therefore been studied in great detail. An improved understanding of these interactions results in the ability to manipulate DNA, proteins and other biomolecules, as well as cells, spatially and temporally at surfaces with high precision. This in turn engenders the development of advanced devices, such as biosensors, bioelectronic components, smart biomaterials and microarrays. Spatial control can be achieved by the production of patterned surface chemistries using modern high-resolution patterning technologies based on lithography, microprinting or microfluidics, whilst temporal control is accessible through the application of switchable surface architectures. The combination of these two surface properties offers unprecedented control over the behaviour of biomolecules and cells at the solid-liquid interface. This review discusses the behaviour of biomolecules and cells at solid interfaces and highlights fundamental and applied research exploring patterned and switchable surfaces.

Dopamine-assisted immobilization of poly(ethylene imine) based polymers for control of cell–surface interactions

Non-fouling coatings play a critical role in many biomedical applications, such as diagnostic assay materials, biosensors, blood contacting devices and other implants. In the present work we have developed a facile, one step deposition method based on dopamine polymerization for preparation of non-fouling and biotinylated surfaces for biomedical applications. Poly(ethylene imine)-graft-poly(ethylene glycol) co-polymer (PEI-g-PEG) was mixed with an alkaline dopamine solution and then deposited onto different substrates. The dopamine coatings formed by this method were characterized by X-ray photoelectron spectroscopy (XPS), and the results indicated successful deposition of PEG. The resultant dopamine coatings formed on tissue culture polystyrene by this method revealed successful deposition of PEG, as shown by XPS. PEI-g-PEG/dopamine deposition for 2h inhibited the adsorption of serum proteins and the attachment of fibroblasts, suggesting that PEG molecules were immobilized in a sufficient density on the surface of the coating. Furthermore, co-deposition of PEI-g-PEG and PEI-g-biotin in alkaline dopamine solutions provided a cell-resisting background surface, at the same time providing accessible biotin molecules. We have demonstrated that the surface can be used for the selective binding of avidin, followed by the binding of Arg-Gly-Asp-Ser-biotin and enhanced cell attachment by specific cell-ligand interactions. In conclusion, our one step immobilization method provides a simple tool to fabricate surfaces with controllable cell affinity.

Clinical observations of biofouling on PEO coated silicone hydrogel contact lenses

Silicone hydrogel contact lenses, which have been a major advance in the field of vision correction, require surface modification or coatings for comfort and biocompatibility. While current coatings show adequate clinical performance, advanced coatings may improve the biocompatibility of contact lenses further by reducing biofouling and related adverse clinical events. Here, we have produced coatings on Lotrafilcon A contact lenses by deposition of a thin film of allylamine plasma polymer (ALAPP) as a reactive interlayer for the high density grafting of poly(ethylene oxide) dialdehyde (PEO(ALD)(2)), which had previously shown complete resistance to protein adsorption in vitro. The performance of these contact lenses was evaluated in a controlled clinical study over 6h using Focus Night and Day (also known as Air Optix Night & Day) contact lenses as control lenses. Surface modified lenses were characterised by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) before and after wear. Clinical data showed a high level of biocompatibility of the PEO coated lenses equivalent to control lenses. Surface analysis of worn contact lenses demonstrated that the high density PEO coating is effective in reducing biofouling in vivo compared to control lenses, however small amounts of protein deposits were still detected on all worn contact lenses. This study highlights that elimination of biofouling in vivo can be much more demanding than in vitro and discusses issues that are important for the analysis of worn contact lenses as well as the design of improved contact lenses.

Nanometer thickness laser ablation for spatial control of cell attachment

We demonstrate here a new method to control the location of cells on surfaces in two dimensions, which can be applied to a number of biomedical applications including diagnostic tests and tissue engineered medical devices. Two-dimensional control over cell attachment is achieved by generation of a spatially controlled surface chemistry that allows control over protein adsorption, a process which mediates cell attachment. Here, we describe the deposition of thin allylamine plasma polymer coatings on silicon wafer and perfluorinated poly(ethylene-co-propylene) substrates, followed by grafting of a protein resistant layer of poly(ethylene oxide). Spatially controlled patterning of the surface chemistry was achieved in a fast, one-step procedure by nanometer thickness controlled laser ablation using a 248 nm excimer laser. X-ray photoelectron spectroscopy and atomic force microscopy were used to confirm the production of surface chemistry patterns with a resolution of approximately 1 µm, which is significantly below the dimensions of a single mammalian cell. Subsequent adsorption of the extracellular matrix proteins collagen I and fibronectin followed by cell culture experiments using bovine corneal epithelial cells confirmed that cell attachment is controlled by the surface chemistry pattern. The method is an effective tool for use in a number of in vitro and in vivo applications.

Advanced Substrate Fabrication for Cell Microarrays

The fabrication and characterization of chemical patterns using a technique that can be readily integrated with methods currently used for the formation of microarrays is presented. A high density poly(ethylene glycol) coating was deposited on glass slides as a background exhibiting low cell attachment properties. Phenylazide modified polymers were then printed on this background. UV irradiation of these polymer arrays resulted in the cross-linking of the polymer spots and their covalent attachment to the surface. Cell attachment was shown to follow the resultant surface chemistry pattern. Furthermore, the use of a robotic contact printer enabled the facile deposition of DNA microarrays on top of and aligned with the polymer microarrays. A transfected cell microarray was generated in this way, demonstrating not only the ability of this platform to limit cell attachment to specific regions, but the suitability for chip-based functional genomics, in particular, and high density cell assays in general.

Combined immunocapture and laser desorption/ionization mass spectrometry on porous silicon.

There is considerable interest in the highly parallelized mass spectrometry analysis of complex sample mixtures without any time-consuming prepurification. Porous silicon-based laser desorption/ionization mass spectrometry (pSi LDI-MS) is enabling technology for such analysis. Previous studies have focused on pSi surface functionalization to enhance sensitivity of detection and engineer surfaces for sample capture and enrichment in LDI-MS analysis. In this report, we build on this work by showing that surface functionalization of thin pSi films can be extended to the covalent immobilization of antibodies, producing a porous immunoaffinity surface. We demonstrate highly selective mass spectrometric detection of illicit drugs (benzodiazepines) on pSi films displaying antibenzodiazepine antibodies covalently immobilized via isocyanate chemistry. The effects of antibody immobilization conditions, antibody concentration, and surface blocking on LDI-MS performance and selectivity were studied. X-ray photoelectron spectroscopy (XPS) was instrumental in characterizing surface chemistry and optimizing LDI-MS performance. Overall, our approach is suitable for rapid and sensitive confirmatory analysis in forensic toxicology requiring only minimal sample volume and may be applied to other areas requiring small molecular analysis such as metabolomics and pharmacology.