Nanoporous Aluminium Oxide Membranes as Cell Interfaces

Nanoporous anodic aluminium oxide (AAO) has become increasingly important in biomedical applications over the past years due to its biocompatibility, increased surface area, and the possibility to tailor this nanomaterial with a wide range of surface modifications. AAO nanopores are formed in an inexpensive anodisation process of pure aluminium, which results in the self-assembly of highly ordered, vertical nanochannels with well-controllable pore diameters, depths, and interpore distances. Because of these outstanding properties AAO nanopores have become excellent candidates as nanostructured substrates for cell-interface studies. In this comprehensive review previous surveys on cell adhesion and proliferation on different AAO nanopore geometries and surface modifications are highlighted and summarised tabularly. Future applications of nanoporous alumina membranes in biotechnology and medicine are also outlined, for instance, the use of nanoporous AAO as implant modifications, coculture substrates, or immunoisolation devices

Nanostrukturierte Metallelektroden zur funktionalen Kopplung an neuronale Zellen

In der modernen biomedizinischen Forschung steigt stetig die Nachfrage nach neuartigen Biosensoren, die die Schnittstelle zwischen physikalischen und biologischen Systemen bilden. Solche bioelektronischen Systeme werden z.B. zur Ableitung extrazellulärer Signale von elektrisch aktiven Zellen oder zur Detektion von DNA eingesetzt. In zunehmendem Maße finden sie auch Verwendung als Neuroimplantate oder Testsysteme für neue pharmazeutische Wirkstoffe. Zur Ableitung extrazellulärer Signale wurden bislang planare Mikroelektroden aus verschiedenen biokompatiblen Metallen verwendet, die seit den 70er Jahren in der Bioelektronik etabliert sind. Um die Qualität der abgeleiteten Signale zu verbessern, wurde das Elektrodendesign stetig weiterentwickelt. Maßgebend für die Signalqualität ist das Signal- Rausch-Verhältnis (SRV), das durch die Schnittstelle zwischen Zelle und Elektrode bestimmt wird. Die Vergrößerung der effektiven Elektrodenoberfläche mit unterschiedlichen porösen Materialien hat bereits zu einer Erhöhung des SRVs geführt. Allerdings zeigten bisherige poröse Elektroden Probleme in der mechanischen Stabilität und der Adhäsion von Zellen und Gewebe. Überdies ist die Herstellung aufwändig und eine Wiederverwendung der Elektroden nur eingeschränkt möglich. Einen neuen, vielversprechenden Lösungsansatz zur Verbesserung der Signalableitung stellt die Modifikation von Mikroelektroden-Arrays (MEAs) mit dreidimensionalen metallischen Nanostrukturen dar. Die Entwicklung und Charakterisierung dieser nanostrukturierten Biochips sowie deren Kopplung an elektrisch aktive Zellen bildeten den Schwerpunkt der vorliegenden Arbeit. Als Material für die Nanostrukturen wurde Gold gewählt, da es aufgrund seiner chemischen und mechanischen Beständigkeit sowie seiner Biokompatiblität für bioelektronische Anwendungen prädestiniert ist. Außerdem besteht über Thiolbindungen die Möglichkeit verschiedener Oberflächenmodifikationen zur gezielten Beeinflussung des Zellwachstums. [...

Nanostrukturierte Metallelektroden zur funktionalen Kopplung an neuronale Zellen

In der modernen biomedizinischen Forschung steigt stetig die Nachfrage nach neuartigen Biosensoren, die die Schnittstelle zwischen physikalischen und biologischen Systemen bilden. Solche bioelektronischen Systeme werden z.B. zur Ableitung extrazellulärer Signale von elektrisch aktiven Zellen oder zur Detektion von DNA eingesetzt. In zunehmendem Maße finden sie auch Verwendung als Neuroimplantate oder Testsysteme für neue pharmazeutische Wirkstoffe. Zur Ableitung extrazellulärer Signale wurden bislang planare Mikroelektroden aus verschiedenen biokompatiblen Metallen verwendet, die seit den 70er Jahren in der Bioelektronik etabliert sind. Um die Qualität der abgeleiteten Signale zu verbessern, wurde das Elektrodendesign stetig weiterentwickelt. Maßgebend für die Signalqualität ist das Signal-Rausch-Verhältnis (SRV), das durch die Schnittstelle zwischen Zelle und Elektrode bestimmt wird. Die Vergrößerung der effektiven Elektrodenoberfläche mit unterschiedlichen porösen Materialien hat bereits zu einer Erhöhung des SRVs geführt. Allerdings zeigten bisherige poröse Elektroden Probleme in der mechanischen Stabilität und der Adhäsion von Zellen und Gewebe. Überdies ist die Herstellung aufwändig und eine Wiederverwendung der Elektroden nur eingeschränkt möglich. Einen neuen, vielversprechenden Lösungsansatz zur Verbesserung der Signalableitung stellt die Modifikation von Mikroelektroden-Arrays (MEAs) mit dreidimensionalen metallischen Nanostrukturen dar. Die Entwicklung und Charakterisierung dieser nanostrukturierten Biochips sowie deren Kopplung an elektrisch aktive Zellen bildeten den Schwerpunkt der vorliegenden Arbeit. Als Material für die Nanostrukturen wurde Gold gewählt, da es aufgrund seiner chemischen und mechanischen Beständigkeit sowie seiner Biokompatiblität für bioelektronische Anwendungen prädestiniert ist. Außerdem besteht über Thiolbindungen die Möglichkeit verschiedener Oberflächenmodifikationen zur gezielten Beeinflussung des Zellwachstums. [...

Model systems for studying cell adhesion and biomimetic actin networks

Many cellular processes, such as migration, proliferation, wound healing and tumor progression are based on cell adhesion. Amongst different cell adhesion molecules, the integrin receptors play a very significant role. Over the past decades the function and signalling of various such integrins have been studied by incorporating the proteins into lipid membranes. These proteolipid structures lay the foundation for the development of artificial cells, which are able to adhere to substrates. To build biomimetic models for studying cell shape and spreading, actin networks can be incorporated into lipid vesicles, too. We here review the mechanisms of integrin-mediated cell adhesion and recent advances in the field of minimal cells towards synthetic adhesion. We focus on reconstituting integrins into lipid structures for mimicking cell adhesion and on the incorporation of actin networks and talin into model cells

Single-molecule mechanics of protein-labelled DNA handles

DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA–protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG)-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp) were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG) beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD) imaging control experiments revealed that quantum dot–streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein–DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular recognition in time-critical molecular motor studies

A nanoporous alumina microelectrode array for functional cell–chip coupling

The design of electrode interfaces has a strong impact on cell-based bioelectronic applications. We present a new type of microelectrode array chip featuring a nanoporous alumina interface. The chip is fabricated in a combination of top-down and bottom-up processes using state-of-the-art clean room technology and self-assembled generation of nanopores by aluminum anodization. The electrode characteristics are investigated in phosphate buffered saline as well as under cell culture conditions. We show that the modified microelectrodes exhibit decreased impedance compared to planar microelectrodes, which is caused by a nanostructuring effect of the underlying gold during anodization. The stability and biocompatibility of the device are demonstrated by measuring action potentials from cardiomyocyte-like cells growing on top of the chip. Cross sections of the cell–surface interface reveal that the cell membrane seals the nanoporous alumina layer without bending into the sub-50 nm apertures. The nanoporous microelectrode array device may be used as a platform for combining extracellular recording of cell activity with stimulating topographical cues

Nanofiber topographies enhance platelet-fibrinogen scaffold interactions.

The initial contact with blood and its components, including plasma proteins and platelets, directs the body's response to foreign materials. Natural scaffolds of extracellular matrix or fibrin contain fibrils with nanoscale dimensions, but how platelets specifically respond to the topography and architecture of fibrous materials is still incompletely understood. In this study, we fabricate planar and nanofiber scaffolds from native fibrinogen to characterize the morphology of adherent platelets and activation markers for phosphatidylserine (PS) exposure and α-granule secretion by confocal fluorescence microscopy and scanning electron microscopy (SEM). Different fibrinogen topographies equally support the spreading and granule secretion of washed platelets. In contrast, preincubation of the scaffolds with plasma diminishes platelet spreading on planar fibrinogen surfaces but not on nanofibers. Our data show that the enhanced interactions of platelets with nanofibers results from a higher locally accessible surface area, effectively increasing the ligand density for integrin-mediated responses. Overall, fibrinogen nanofibers direct platelets towards robust adhesion formation and α-granule secretion while minimizing their pro-coagulant activity. Similar results on fibrinogen-coated PDMS substrates with micron-sized 3D features suggest that surface topography could be used more generally to steer blood-materials interactions on different length scales for enhancing the initial wound healing steps.</p

Controlling the Multiscale Structure of Nanofibrous Fibrinogen Scaffolds for Wound Healing

As a key player in blood coagulation and tissue repair, fibrinogen has gained increasing attention to develop nanofibrous biomaterial scaffolds for wound healing. Current techniques to prepare protein nanofibers, like electrospinning or extrusion, are known to induce lasting changes in the protein conformation. Often, such secondary changes are associated with amyloid transitions, which can evoke unwanted disease mechanisms. Starting from our recently introduced technique to self-assemble fibrinogen scaffolds in physiological salt buffers, we here investigated the morphology and secondary structure of our novel fibrinogen nanofibers. Aiming at optimum self-assembly conditions for wound healing scaffolds, we studied the influence of fibrinogen concentration and pH on the protein conformation. Using circular dichroism and Fourier-transform infrared spectroscopy, we observed partial transitions from α-helical structures to β-strands upon fiber formation. Interestingly, a staining with thioflavin T revealed that this conformational transition was not associated with any amyloid formation. Toward novel scaffolds for wound healing, which are stable in aqueous environment, we also introduced cross-linking of fibrinogen scaffolds in formaldehyde vapor. This treatment allowed us to maintain the nanofibrous morphology while the conformation of fibrinogen nanofibers was redeveloped toward a more native state after rehydration. Altogether, self-assembled fibrinogen scaffolds are excellent candidates for novel wound healing systems since their multiscale structures can be well controlled without inducing any pathogenic amyloid transitions